U.S. patent application number 10/269557 was filed with the patent office on 2003-05-29 for heat shock genes and proteins from neisseria meningitidis, candida glabrata and aspergillus fumigatus.
This patent application is currently assigned to Stressgen Biotechnologies Corporation, a Victoria, Canada corporation. Invention is credited to Wisniewski, Jan.
Application Number | 20030099664 10/269557 |
Document ID | / |
Family ID | 22770344 |
Filed Date | 2003-05-29 |
United States Patent
Application |
20030099664 |
Kind Code |
A1 |
Wisniewski, Jan |
May 29, 2003 |
Heat shock genes and proteins from Neisseria meningitidis, Candida
glabrata and Aspergillus fumigatus
Abstract
Methods and compositions comprising isolated nucleic acid
molecules specific to Neisseria meningitidis, Candida glabrata and
Aspergillus fumigatus heat shock proteins (Hsps), as well as vector
constructs and isolated polypeptides specific to the same are
provided. Such compositions and methods are useful for the
diagnosis of infections by these organisms and for generating an
immune response to the organisms.
Inventors: |
Wisniewski, Jan; (Sooke,
CA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
Stressgen Biotechnologies
Corporation, a Victoria, Canada corporation
|
Family ID: |
22770344 |
Appl. No.: |
10/269557 |
Filed: |
October 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10269557 |
Oct 11, 2002 |
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09207388 |
Dec 8, 1998 |
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6497880 |
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Current U.S.
Class: |
424/190.1 ;
435/252.3; 435/320.1; 435/6.12; 435/69.3; 530/350; 536/23.7 |
Current CPC
Class: |
C07K 2319/00 20130101;
G01N 33/56911 20130101; C07K 14/38 20130101; G01N 33/56961
20130101; C07K 14/22 20130101; G01N 2333/38 20130101; A61K 39/00
20130101; C07K 14/40 20130101; G01N 2333/40 20130101; A61K 38/00
20130101; G01N 2333/22 20130101; G01N 2469/20 20130101 |
Class at
Publication: |
424/190.1 ;
435/6; 435/69.3; 435/252.3; 435/320.1; 536/23.7; 530/350 |
International
Class: |
A61K 039/02; C12Q
001/68; C07H 021/04; C12P 021/02; C12N 005/06; C07K 014/22 |
Claims
1. An isolated nucleic acid molecule encoding a Neisseria
meningitidis Hsp70.
2. An isolated nucleotide molecule selected from the group
consisting of: (a) an isolated nucleic acid molecule comprising the
sequence of SEQ ID NO: 1; (b) an isolated nucleic acid molecule
comprising the sequence of SEQ ID NO: 1 from nucleotides 358-2286;
(c) an isolated nucleic acid molecule comprising the sequence of
SEQ ID NO: 2 from nucleotides 4-1932; (d) an isolated nucleic acid
molecule comprising the sequence of SEQ ID NO: 3; (e) an isolated
nucleic acid molecule comprising the sequence of SEQ ID NO: 4; and
(f) an isolated nucleic acid molecule comprising a sequence
complementary to any one of the isolated nucleotide molecules set
forth in (a) through (e), respectively:
3. An isolated nucleic acid molecule which is a variant of, or is
substantially similar to, the nucleotide molecule according to
claim 2.
4. An isolated nucleic acid molecule comprising a nucleotide
sequence that is identical to a segment of contiguous nucleotide
bases comprising at least 25% of any one of: SEQ. ID NO: 1 from
nucleotides 358-2286; SEQ. ID NO: 2 from nucleotides 4-1932; SEQ ID
NO: 3; SEQ. ID NO: 4; or to a complement thereof.
5. An isolated nucleic acid molecule comprising a nucleic acid
sequence that encodes a polypeptide comprising any one of the
polypeptides according to FIGS. 2, 4, 6, 8, or 9 or a variant Hsp70
that is at least 95% homologous to a polypeptide according to any
one of FIGS. 2, 4, 6, 8, or 9, wherein percent homology is
determined according to an algorithm incorporated in a protein
database search program used in BLAST.TM. or DNA Star
Megalign.TM..
6. The isolated nucleic acid molecule according to claim 5 wherein
the encoded polypeptide is able to selectively bind an antibody
specific for a Neisseria meningitidis Hsp70.
7. An isolated nucleic acid molecule encoding at least 8 contiguous
amino acids of a Neisseria meningitis Hsp70 polypeptide selected
from amino acid residues of FIG. 6, wherein the encoded Neisseria
meningitidis Hsp70 polypeptide is able to bind to a major
histocompatibility complex.
8. An isolated Neisseria meningitidis Hsp70 polypeptide.
9. An isolated Hsp70 polypeptide comprising the amino acid sequence
of according to FIG. 6 or variants thereof.
10. An isolated Hsp70 polypeptide that is able to selectively bind
to an antibody specific for a Neisseria meningitidis Hsp70.
11. The isolated Hsp70 polypeptide according to claim 9 wherein the
isolated Hsp70 polypeptide is fused to an additional polypeptide to
create a fusion protein.
12. An isolated Hsp70 polypeptide comprising at least 8 contiguous
amino acids selected from amino acid residues of FIG. 6, wherein
the Hsp70 polypeptide is able to bind to a major histocompatibility
complex.
13. The isolated Hsp70 polypeptide according to claim 12 wherein
binding to the major histocompatibility complex elicits or enhances
an immune response to Neisseria meningitidis in a human being.
14. The isolated Hsp70 polypeptide according to claim 12, wherein
the Hsp70 polypeptide is obtained by proteolytic cleavage.
15. The isolated Hsp70 polypeptide according to claim 12, wherein
the Hsp70 polypeptide is obtained by chemical synthesis.
16. The isolated Hsp70 polypeptide according to claim 12, wherein
the Hsp70 polypeptide is obtained by expression in a transformed
host cell containing a nucleic acid molecule encoding the Hsp70
polypeptide or portion thereof.
17. An isolated Hsp70 polypeptide comprising an amino acid sequence
having at least 95% homology to the Hsp70 polypeptide of FIG. 6 and
which is able to selectively bind to an antibody specific for a
Neisseria meningitidis Hsp70, wherein percent homology is
determined according to an algorithm incorporated in a protein
database search program used in BLAST.TM. or DNA Star
Megalign.TM..
18. An isolated Hsp70 polypeptide wherein the polypeptide is an
expression product of a transformed host cell containing an
isolated nucleic acid molecule according to any one of claims
1-7.
19. A vector containing an isolated nucleic acid molecule according
to any one of claims 1-7.
20. The vector according to claim 19 wherein the vector is an
expression vector.
21. The vector according to claim 20 further comprising a
selectable or identifiable marker and wherein the promoter is a
constitutive or an inducible promoter.
22. A host cell containing a vector according to claim 19.
23. The host cell of claim 22 wherein the host cell is selected
from the group consisting of a bacterial cell, a mammalian cell, a
yeast cell, a plant cell and an insect cell.
24. A composition comprising an Hsp70 polypeptide according to any
one of claims 8-17 in combination with a pharmaceutically
acceptable carrier or diluent.
25. A composition comprising a Hsp70polypeptide according to claim
18 in combination with a pharmaceutically acceptable carrier or
diluent.
26. The composition of claim 24 wherein the pharmaceutically
acceptable carrier or diluent is suitable for at least one of:
systemic administration, oral administration, intranasal
administration or parenteral administration.
27. The composition of claim 25 wherein the pharmaceutically
acceptable carrier or diluent is suitable for at least one of:
systemic administration, oral administration, intranasal
administration or parenteral administration.
28. A method for eliciting or enhancing an immune response in a
mammal against Neisseria meningitidis, comprising administering to
the mammal in an amount effective to elicit or enhance the
response, an Hsp70 polypeptide according to any one of claims 8-17
in combination with a pharmaceutically acceptable carrier or
diluent.
29. A method for eliciting or enhancing an immune response in a
mammal against Neisseria meningitidis, comprising administering to
the mammal in an amount effective to elicit or enhance the
response, an Hsp70 polypeptide according to claim 18 in combination
with a pharmaceutically acceptable carrier or diluent.
30. A method for eliciting or enhancing an immune response in a
mammal against a target antigen comprising administering to the
mammal the target antigen joined to an Hsp70 polypeptide according
to any one of claims 8-17 in combination with a pharmaceutically
acceptable carrier or diluent.
31. A method for eliciting or enhancing an immune response in a
mammal against a target antigen comprising administering to the
mammal the target antigen joined to an Hsp70 polypeptide according
to claim 18 in combination with a pharmaceutically acceptable
carrier or diluent.
32. A method for eliciting or enhancing an immune response in a
mammal to a polypeptide comprising administering to the mammal a
fusion protein containing sequences of the polypeptide fused to
sequences of an Neisseria meningitidis Hsp70 polypeptide in
combination with a pharmaceutically acceptable carrier or
diluent.
33. A probe or PCR primer for detecting DNA encoding a Neisseria
meningitidis Hsp70 comprising at least about 15 contiguous bases
from any one of SEQ. ID NOS: 1-4, or a compliment thereof.
34. A method for diagnosing the presence of a Neisseria
meningitidis in a subject sample comprising: obtaining a DNA
fraction from the subject sample; and performing a PCR
amplification of the DNA fraction using at least one PCR primer
comprised of at least about 15 contiguous bases from any one of
SEQ. ID NOS: 1-4, or a compliment thereof.
35. An isolated nucleic acid molecule encoding a Aspergillus
fumigatus Hsp60 polypeptide.
36. An isolated nucleic acid molecule selected from the group
consisting of: (a) an isolated nucleic acid molecule comprising the
sequence of SEQ ID NO: 5; (b) an isolated nucleic acid molecule
comprising the sequence of SEQ ID NO: 5 from nucleotides 300-2234;
(c) an isolated nucleic acid molecule comprising the sequence of
SEQ ID NO: 5 from nucleotides 300-410. nucleotides 514-655 and
nucleotides 724-2234; (d) an isolated nucleic acid molecule
comprising-the sequence of SEQ ID NO: 6; (e) an isolated nucleic
acid molecule comprising the sequence of SEQ ID NO: 7; (f) an
isolated nucleic acid molecule complementary to any one of the
nucleotides of SEQ ID NOS: 5 to 7 set forth in (a) through (e),
respectively.
37. An isolated nucleic acid molecule which is a variant of, or is
substantially similar to, the nucleotide molecule according to
claim 36.
38. An isolated nucleic acid molecule comprising a nucleotide
sequence that is identical to a segment of contiguous nucleotide
bases comprising at least 25% of any one of SEQ ID NOS: 5-7; or to
a complement thereof.
39. An isolated nucleic acid molecule encoding Hsp60 comprising a
nucleic acid sequence that encodes a polypeptide comprising any one
of the polypeptides according to FIGS. 14, 16 or 18; or a variant
Hsp60 that is at least 95% homologous to a polypeptide according to
any one of FIGS. 14, 16 or 18, wherein percent homology is
determined according to an algorithm incorporated in a protein
database search program used in BLAST.TM. or DNA Star
Megalign.TM..
40. The isolated nucleic acid molecule according to claim 39
wherein the encoded polypeptide is able to selectively bind an
antibody specific for a Aspergillus fumigatus Hsp60.
41. An isolated nucleic acid molecule encoding at least 8
contiguous amino acids of an Aspergillus fumigatus Hsp60
polypeptide selected from amino acid residues according to FIG. 14,
wherein the encoded Aspergillus fumigatus Hsp60 polypeptide is able
to bind a major histocompatibility complex.
42. An isolated Aspergillus fumigatus Hsp60 polypeptide.
43. An isolated Hsp60 polypeptide comprising the amino acid
sequence according to FIG. 14, or variants thereof.
44. An isolated Hsp60 polypeptide that is able to selectively bind
to an antibody specific for a Aspergillus fumigatus Hsp60.
45. The isolated Hsp60 polypeptide according to claim 43 wherein
the isolated Hsp60 polypeptide is fused to an additional
polypeptide to create a fusion protein.
46. An isolated Hsp70 polypeptide comprising at least 8 contiguous
amino acids selected from amino acid residues selected from amino
acid residues of FIG. 14, wherein the Hsp60 polypeptide is able to
bind to a major histocompatibility complex.
47. The isolated Hsp60 polypeptide according to claim 46 wherein
binding to the major histocompatibility complex elicits or enhances
an immune response to Aspergillus fumigatus in a human being.
48. The isolated Hsp60 polypeptide according to claim 46, wherein
the Hsp60 polypeptide is obtained by proteolytic cleavage
49. The isolated Hsp60 polypeptide according to claim 46, wherein
the Hsp60 polypeptide is obtained by chemical synthesis.
50. The isolated Hsp60 polypeptide according to claim 46, wherein
the Hsp60 polypeptide is obtained by expression in a transformed
host cell containing a nucleic acid molecule encoding the Hsp60
polypeptide or portion thereof.
51. An isolated Hsp60 polypeptide comprising an amino acid sequence
having at least 95% homology to the Hsp60 polypeptide of FIG. 14,
and which is able to selectively bind to an antibody specific for
an Aspergillus fumigatus Hsp60, wherein percent homology is
determined according to an algorithm incorporated in a protein
database search program used in BLAST.TM. or DNA Star
Megalign.TM..
52. An isolated Hsp60 polypeptide wherein the polypeptide is an
expression product of a transformed host cell containing an
isolated nucleic acid molecule according to any one of claims
35-41.
53. A vector containing an isolated nucleic acid molecule according
to any one of claims 35-41.
54. The vector according to claim 53 wherein the vector is an
expression vector.
55. The vector according to claim 54 further comprising a
selectable or identifiable marker and wherein the promoter is a
constitutive or an inducible promoter.
56. A host cell containing a vector according to claim 53.
57. The host cell according to claim 56 wherein the host cell is
selected from the group consisting of a bacterial cell, a mammalian
cell, a yeast cell, a plant cell and an insect cell.
58. A composition comprising an Hsp60 polypeptide according to any
one of claims 35-41 in combination with a pharmaceutically
acceptable carrier or diluent.
59. A composition comprising a Hsp60 polypeptide according to claim
47 in combination with a pharmaceutically acceptable carrier or
diluent.
60. The composition of claim 58 wherein the pharmaceutically
acceptable carrier or diluent is suitable for at least one of:
systemic administration, oral administration, intranasal
administration or parenteral administration.
61. The composition of claim 59 wherein the pharmaceutically
acceptable carrier or diluent is suitable for at least one of:
systemic administration, oral administration, intranasal
administration or parenteral administration.
62. A method for eliciting or enhancing an immune response in a
mammal against Aspergillus fumigatus, comprising administering to
the mammal in an amount effective to elicit or enhance the
response, an Hsp60 polypeptide according to any one of claims 35-41
in combination with a pharmaceutically acceptable carrier or
diluent.
63. A method for eliciting or enhancing an immune response in a
mammal against Aspergillus fumigatus, comprising administering to
the mammal in an amount effective to elicit or enhance the
response, an Hsp60 polypeptide according to claim 47 in combination
with a pharmaceutically acceptable carrier or diluent.
64. A method for eliciting or enhancing an immune response in a
mammal against a target antigen comprising administering to the
mammal the target antigen joined to an Hsp60 polypeptide according
to any one of claims 35-41 in combination with a pharmaceutically
acceptable carrier or diluent.
65. A method for eliciting or enhancing an immune response in a
mammal against a target antigen comprising administering to the
mammal the target antigen joined to an Hsp60 polypeptide according
to claim 47 in combination with a pharmaceutically acceptable
carrier or diluent.
66. A method for eliciting or enhancing an immune response in a
mammal to a polypeptide comprising administering to the mammal a
fusion protein containing sequences of the polypeptide fused to
sequences of an Aspergillus fumigatus Hsp60 polypeptide in
combination with a pharmaceutically acceptable carrier or
diluent.
67. A probe or PCR primer for detecting DNA encoding a Aspergillus
fumigatus Hsp60 comprising at least about 15 contiguous bases from
any one of SEQ. ID NOS: 5-7, or a compliment thereof.
68. A method for diagnosing the presence of a Aspergillus fumigatus
in a subject sample comprising: obtaining a DNA fraction from the
subject sample; and performing a PCR amplification of the DNA
fraction using at least one PCR primer comprised of at least about
15 contiguous bases from any one of SEQ. ID NOS: 5-7, or a
compliment thereof.
69. An isolated nucleic acid molecule encoding a Candida glabrata
Hsp60.
70. An isolated nucleotide molecule selected from the group
consisting of: (a) an isolated nucleic acid molecule comprising the
sequence of SEQ ID NO: 8; (b) an isolated nucleic acid molecule
comprising the sequence of SEQ ID NO: 8 from nucleotides 258-1964;
(c) an isolated nucleic acid molecule comprising the sequence of
SEQ ID NO: 9; (d) an isolated nucleic acid molecule comprising the
sequence of SEQ ID NO: 10; (e) an isolated nucleic acid molecule
complementary to any one of the nucleotides of SEQ ID NOS: 8 to 10
set forth in (a) through (d), respectively.
71. An isolated nucleic acid molecule which is a variant of, or is
substantially similar to, the nucleotide molecule according to
claim 70.
72. An isolated nucleic acid comprising a nucleotide sequence that
is identical to a segment of contiguous nucleotide bases comprising
at least 25% of any one of SEQ ID NOS: 8-10, or to a complement
thereof.
73. An isolated nucleic acid molecule encoding Hsp60 comprising a
nucleic acid sequence that encodes a polypeptide comprising any one
of the polypeptides according to FIGS. 21, 23, or 25, or a variant
Hsp60 that is at least 95% homologous to a polypeptide according to
any one of FIGS. 21, 23, or 25, wherein percent homology is
determined according to an algorithm incorporated in a protein
database search program used in BLAST.TM. or DNA Star
Megalign.TM..
74. The isolated nucleic acid molecule according to claim 73
wherein the encoded polypeptide is able to selectively bind an
antibody specific for a Candida glabrata Hsp60.
75. An isolated nucleic acid molecule encoding at least 8
contiguous amino acids of a Candida glabrata Hsp60 polypeptide
selected from amino acid residues according to FIG. 21, wherein the
encoded Candida glabrata Hsp60 polypeptide is able to bind to a
major histocompatibility complex.
76. An isolated Candida glabrata Hsp60 polypeptide.
77. An isolated Hsp60 polypeptide comprising the amino acid
sequence of FIG. 21, or variants thereof.
78. An isolated Hsp60 polypeptide the that is able to selectively
bind to an antibody specific for a Candida glabrata Hsp60.
79. The isolated Hsp60 polypeptide according to claim 77 wherein
the isolated Hsp70 polypeptide is fused to an additional
polypeptide to create a fusion protein.
80. An isolated Hsp60 polypeptide comprising at least 8 contiguous
amino amino acids selected from amino acid residues according to
FIG. 21, wherein the Hsp60 polypeptide is is able to bind to a
major histocompatibility complex.
81. The isolated Hsp60 polypeptide according to claim 80 wherein
binding to the major histocompatibility complex elicits or enhances
an immune response to Candida glabrata in a human being.
82. The isolated Hsp60 polypeptide according to claim 80 wherein
the Hsp60 polypeptide is obtained by proteolytic cleavage.
83. The isolated Hsp60 polypeptide according to claim 80, wherein
the Hsp70 polypeptide is obtained by chemical synthesis.
84. The isolated Hsp60 polypeptide according to claim 80, wherein
the Hsp70 polypeptide is obtained by expression in a transformed
host cell containing a nucleic acid molecule encoding the Hsp60
polypeptide or portion thereof.
85. An isolated Hsp60 polypeptide comprising an amino acid sequence
having at least 95% homology to the Hsp60 polypeptide of FIG. 21,
and which is able to selectively bind to an antibody specific for a
Candida glabrata Hsp60, wherein percent homology is determined
according to an algorithm incorporated in a protein database search
program used in BLAST.TM. or DNA Star Megalign.TM..
86. An isolated Hsp60 polypeptide wherein the polypeptide is an
expression product of a transformed host cell containing an
isolated nucleic acid molecule according to any one of claims
69-75.
87. A vector containing an isolated nucleic acid molecule according
to any one of claims 69-75.
88. The vector according to claim 87 wherein the vector is an
expression vector.
89. The vector according to claim 88 further comprising a
selectable or identifiable marker and wherein the promoter is a
constitutive or an inducible promoter.
90. A host cell containing a vector according to claim 87.
91. The host cell of claim 90 wherein the host cell is selected
from the group consisting of a bacterial cell, a mammalian cell, a
yeast cell, a plant cell and an insect cell.
92. A composition comprising an Hsp60 polypeptide according to any
one of claims 76-85 in combination with a pharmaceutically
acceptable carrier or diluent.
93. A composition comprising a Hsp60 polypeptide according to claim
86 in combination with a pharmaceutically acceptable carrier or
diluent.
94. The composition of claim 92 wherein the pharmaceutically
acceptable carrier or diluent is suitable for at least one of:
systemic administration, oral administration, intranasal
administration or parenteral administration.
95. The composition of claim 92 wherein the pharmaceutically
acceptable carrier or diluent is suitable for at least one of:
systemic administration, oral administration, intranasal
administration or parenteral administration.
96. A method for eliciting or enhancing an immune response in a
mammal against Candida glabrata, comprising administering to the
mammal an in an amount effective to elicit or enhance the response,
an Hsp60 polypeptide according to any one of claims 76-85 in
combination with a pharmaceutically acceptable carrier or
diluent.
97. A method for eliciting or enhancing an immune response in a
mammal against Candida glabrata, comprising administering to the
mammal an in an amount effective to elicit or enhance the response,
an Hsp60 polypeptide according to claim 86 in combination with a
pharmaceutically acceptable carrier or diluent.
98. A method for eliciting or enhancing an immune response in a
mammal against a target antigen comprising administering to the
mammal the target antigen joined to an Hsp60 polypeptide according
to any one of claims 76-85 in combination with a pharmaceutically
acceptable carrier or diluent.
99. A method for eliciting or enhancing an immune response in a
mammal against a target antigen comprising administering to the
mammal the target antigen joined to an Hsp70 polypeptide according
to claim 86 in combination with a pharmaceutically acceptable
carrier or diluent.
100. A method for eliciting or enhancing an immune response in a
mammal to a polypeptide comprising administering to the mammal a
fusion protein containing sequences of the polypeptide fused to
sequences of an Candida glabrata Hsp60 polypeptide in combination
with a pharmaceutically acceptable carrier or diluent.
101. A probe or PCR primer for detecting DNA encoding a Candida
glabrata Hsp60 comprising at least about 15contiguous bases from
any one of SEQ. ID NOS: 8-10, or a compliment thereof.
102. A method for diagnosing the presence of Candida glabrata in a
subject sample comprising: obtaining a DNA fraction from the
subject sample; and performing a PCR amplification of the DNA
fraction using at least one PCR primer comprised of at least about
15 contiguous bases from any one of SEQ. ID NOS: 8-10, or a
compliment thereof.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] This invention relates to heat shock proteins of the Hsp60
family from Candida glabrata and Aspergillus fumigatus and a heat
shock protein of the Hsp70 family from Neisseria meningitidis,
including fragments thereof, and uses of such proteins and nucleic
acid molecules encoding these proteins.
BACKGROUND OF THE INVENTION
[0002] Meningitis is an infection of the fluid of the spinal cord
and the fluid that surrounds the brain. The disease is caused
either by a viral or a bacterial infection. Viral meningitis is
typically less severe than bacterial meningitis and resolves
without specific treatment. In contrast, bacterial meningitis can
be rather severe and can cause brain damage, hearing loss or
learning disability. Symptoms of meningitis are high fever,
headache and stiff neck. These symptoms may develop over a span of
several hours, or may take 1-2 days. Other symptoms may be nausea,
vomiting, discomfort looking into bright light, confusion or
sleepiness. In young children, the classical symptoms may be absent
or may not be easily detected, and the child may appear to be slow,
inactive, irritable, vomiting or feeding poorly. As the disease
progresses, seizures may occur.
[0003] Bacterial meningitis may be caused by Haemophilus
influenzae, Streptococcus pneumoniae or Neisseria meningitidis.
Because all children (in the U.S.) are now given a vaccine against
Haemophilus influenzae in the course of their routine
immunizations, meningitis due to this organism is now relatively
uncommon. Thus, the major bacterial disease-causing agents are now
Streptococcus pneumoniae and Neisseria meningitidis.
[0004] Early diagnosis and treatment are critically important. It
must be determined whether symptoms are due to a viral or bacterial
agents, and, if they are caused by a bacterial agent, which
bacterium is involved. Present methods of diagnosis are relatively
slow. They involve obtaining spinal fluid by performing a Bacteria
are identified by cultivation of spinal fluid.
[0005] Bacterially caused meningitis can be treated by antibiotics.
However is critically important that treatment commence early in
the course of the disease Obviously, antibiotic therapy may be
jeopardized by the development of antibiotic-resistant strains of
disease-causing bacteria. Because of this concern and also because
of cost-benefit considerations, vaccination against the bacteria
causing the disease would be preferable, at least in regions, in
which the disease is endemic. As discussed before, U.S. children
are routinely vaccinated against Haemophilus influenzae, but not
against Neisseria meningitidis and Streptococcus pneumoniae. It is
noted that vaccines against the latter two organisms have been
generated. One such vaccine protects against four strains of
Neisseria meningitidis. However, the vaccine appears not to be
effective in children under 18 months of age. Similarly, a vaccine
containing polysaccharide antigens for 14 of the 83 capsular types
of Streptococcus pneumoniae was developed. The vaccine was found to
be 57% effective in two large studies. As with the Neisseria
vaccine, children under the age of two years do not appear to be
protected by the vaccine. Thus, there is a need for improved
vaccines against the latter two species of bacteria. The
information provided here was obtained from publications by the
Division of Bacterial and Mycotic Diseases of the National Center
for Infectious Diseases, and the Centers for Disease Control and
Prevention (www.cdc.gov/ncidod/dbmd/bactmen.htm; May 28, 1998), by
Lonks and Medeiros, Antimicrobial Therapy 1 79:523-35, 1995, by
Butler et al., JAMA 270:1826, 1993, and by Gotschlich et al.,
Antibodies in Human Diagnosis and Therapy 391-402 (Haber and Krause
eds., 1977).
[0006] Aspergillosis is an opportunistic infection occurring in
compromised individuals and is caused by molds of the genus
Aspergillus, of which Aspergillus fumigatus is an important
species. Aspergillus is ubiquitous and is distributed worldwide.
Infection generally involves inhalation of fungal elements.
Aspergillosis has several clinical manifestations, including
colonization of the ear or the lungs, allergic pulmonary
involvement, and invasive pulmonary and disseminated infections.
The pulmonary invasive and disseminated forms of infections have a
grave prognosis, including a high rate of mortality. Susceptible
hosts include cancer patients, patients treated with
immunosuppressive or cytotoxic drugs, and those otherwise
debilitated such as AIDS patients, neutropenic cancer patients, or
patients receiving adrenal corticosteroid drugs. Segal, Vaccines
against Fungal Infections, CRC Crit. Rev. Microbiology 14:229,
1987. Rolston and Bodey, Infections in patients with cancer, Cancer
Medicine (Holland et al. eds), 1997. The true incidence of
aspergillosis is not known in the HIV/AIDS population, in part
because the condition is frequently not diagnosed. However, it is
clear that the incidence is increasing. Ampel has reported that
more than 75 cases have been documented in the literature. Ampel,
Emerging disease issues and fungal pathogens associated with HIV
infection, Emerging Infectious Diseases 2:109-116, 1996.
Aspergillosis has been reported to occur in 20-50% of patients with
acute leukemia. It is noted that many cases of Aspergillus
infection in cancer patients are not diagnosed until an autopsy is
performed after death. Clearly, aspergillosis is becoming
increasingly common among neutropenic patients and in cancer
patients receiving corticosteroid drugs. Rolston and Bodey, supra.
An article in 1992 by Bodey et al. reports on the incidence of
fungal infections based on an international autopsy survey. Bodey
et al., Fungal Infections In Cancer Patients, An International
Autopsy Survey, Eur. J. Clin. Microbiol. Infect. Dis. 11:99-109,
1992. Countries included are Austria, Belgium, Canada, Germany,
Italy, Japan, Netherlands and the UK. It was concluded that 25% of
leukemia patients had fungal infections. Of these infected
patients, 66% had candidiasis, and 34% aspergillosis. Estimates of
rates of fungal infections in organ transplant patients as high as
about 40% were published. Paya, Clin. Infect. Dis. 16:677-688,
1993. More than 80% of these infections were due to Candida and
Aspergillus. As alluded to before, diagnosis of aspergillosis is
not infrequently missed, and there is therefore a need for improved
methods of diagnosis.
[0007] Candidiasis is a fungal infection caused by yeasts of the
genus Candida. Among the more than 80 known species, only seven
species appear to be pathogenic. The major disease-causing species
is Candida albicans. Among the pathogenic species is also found
Candida glabrata, formerly known as Torulopsis glabrata. Clinical
manifestations of Candida infection range from superficial
cutaneous infections to disseminated disease. Infections range from
acute to chronic. They can involve skin and nails, the mucosal
membranes of the mouth and vagina, and various internal organs such
as the lungs, gastrointestinal tract, and circulatory and central
nervous systems. Manifestations can be oral thrush, vaginitis,
balanitis, diaper rash, chronic mucocutaneous conditions,
bronchitis or pneumonia, meningitis, endocarditis, and septicemia.
While the superficial forms of candidiasis have been well known
since antiquity, the incidence of the disseminated forms has
increased recently, presumably because of the extensive use of
antibiotics, corticosteroids, cytotoxic drugs, organ
transplantation and other complex surgical procedures. It is
important to note that today the majority of systemic or invasive
fungal infections are due to Candida species. Segal, supra.
Stringer, Mass. High Tech. 14:3, 1997.
[0008] Mortality from systemic candidiasis remains high, in the
order of 38-59%. The mainstay for treatment is amphotericin B, and
an alternative is fluconazole. In a multicenter trial, amphotericin
B was 79% effective, and fluconazole 70%. Note that Candida
glabrata is resistant to fluconazole. Because mortality remains
high, an effective vaccine against candidiasis to be used in
high-risk populations would be desirable.
[0009] Since Candida albicans is the major cause of candidiasis,
essentially all work relating to both diagnosis and vaccination
concerned this particular species. Thus, it is not clear to what
extent diagnostic procedures developed and vaccination approaches
taken would also detect or protect against other species such as
Candida glabrata.
[0010] Therefore, there is a need in the art to identify and
isolate novel stress proteins and nucleic acids encoding the same
from Neisseria meningitidis, Candida glabrata and Aspergillus
fumigatus, which are useful in the detection, diagnosis and
treatment of infections caused by these organisms.
SUMMARY OF THE INVENTION
[0011] The present invention provides methods and compositions
comprising isolated nucleic acid molecules specific to Neisseria
meningitidis, Candida glabrata and Aspergillus fumigatus, as well
as vector constructs and isolated polypeptides specific to
Neisseria meningitidis, Candida glabrata and Aspergillus fumigatus.
Such compositions and methods are useful for the diagnosis of
infection and for generating an immune response to the respective
organisms.
[0012] Thus, in one aspect the present invention provides an
isolated nucleic acid molecule encoding a Neisseria meningitidis
Hsp70. In some embodiments, the isolated nucleotide molecule is
selected from the group consisting of: (a) an isolated nucleic acid
molecule comprising the sequence of SEQ ID NO: 1 (FIG. 4); (b) an
isolated nucleic acid molecule comprising the sequence of SEQ ID
NO: 1 from nucleotides 358-2286; (c) an isolated nucleic acid
molecule comprising the sequence of SEQ ID NO: 2 (FIG. 6) from
nucleotides 4-1932; (d) an isolated nucleic acid molecule
comprising the sequence of SEQ ID NO: 3 (FIG. 8); (e) an isolated
nucleic acid molecule comprising the sequence of SEQ ID NO: 4 (FIG.
9); (f) an isolated nucleic acid molecule complementary to any one
of the nucleotides of SEQ ID NOS: 1 to 4 set forth in (a) through
(e), respectively.
[0013] In another aspect, the present invention provides an
isolated nucleic acid molecule which is a variant of, or is
substantially similar to, the Neisseria Hsp70 nucleotide molecules
described above. In further aspects the present invention provides
an isolated nucleic acid molecule comprising a nucleotide sequence
that is identical to a segment of contiguous nucleotide bases
comprising at least 25% of SEQ ID NOS: 1 to 4 or a complement
thereof or an isolated nucleic acid molecule encoding Hsp70
comprising a nucleic acid sequence that encodes a polypeptide
comprising any one of SEQ ID NOS: 2, 3, or 4 or a variant Hsp70
that is at least 95% homologous to a polypeptide according to any
one of SEQ ID NOS: 2, 3, or 4.
[0014] In one embodiment, the present invention provides an
isolated nucleic acid molecule as described above, the molecule
encoding a polypeptide that is able to be selectively bound by an
antibody specific for a Neisseria meningitidis Hsp70.
[0015] In still another aspect, the present invention provides an
isolated nucleic acid molecule encoding at least 8 contiguous amino
acids of a Neisseria meningitis Hsp70 polypeptide selected from
amino acid residues of FIG. 6, wherein the encoded Neisseria
meningitidis Hsp70 polypeptide is able to bind to a major
histocompatibility complex.
[0016] In still further aspects the present invention provides an
isolated Neisseria meningitidis Hsp70 polypeptide.
[0017] In some embodiments, the isolated Hsp70 polypeptide
comprises the amino acid sequence of FIG. 6, or variants thereof,
preferably wherein the polypeptide is able to be selectively bound
by an antibody specific for a Neisseria meningitidis Hsp70. In
further embodiments, the isolated Hsp70 polypeptide is fused to an
additional polypeptide to create a fusion protein.
[0018] In still yet further aspects the present invention provides
an isolated Hsp70 polypeptide comprising at least 8 contiguous
amino acids selected from amino acid residues of FIG. 6, wherein
the Hsp70 polypeptide is capable of binding to a major
histocompatibility complex and eliciting or enhancing an immune
response to Neisseria meningitidis in a human being.
[0019] In certain embodiments, the isolated Hsp70 polypeptide is
derived by proteolytic cleavage or chemical synthesis, or is an
expression product of a transformed host cell containing a nucleic
acid molecule encoding the Hsp70 or portion thereof. In further
certain embodiments, the isolated Hsp70 polypeptide comprises
greater than 95% homology to the Hsp70 polypeptide of FIG. 6, and
the isolated Hsp70 polypeptide is able to be selectively bound by
an antibody specific for a Neisseria meningitidis Hsp70.
[0020] In still yet another aspect the present invention provides
an isolated polypeptide wherein the polypeptide is an expression
product of a transformed host cell containing one of the
aforementioned nucleic acid molecules derived from Neisseria.
[0021] In still yet further aspects the present invention provides
vectors comprising at least one of the aforementioned nucleic acid
molecules derived from Neisseria. In certain embodiments, the
vector is an expression vector comprising a promoter in operative
linkage with the isolated nucleic acid molecule encoding the Hsp70
or portion thereof, preferably further comprising a selectable or
identifiable marker and/or wherein the promoter is a constitutive
or an inducible promoter. The present invention also provides host
cells containing such vectors. In certain embodiments, the host
cell is selected from the group consisting of a bacterial cell, a
mammalian cell, a yeast cell, a plant cell and an insect cell.
[0022] In still yet other aspects the present invention provides
compositions comprising a Neisseria Hsp70 polypeptide in
combination with a pharmaceutically acceptable carrier or diluent.
In certain embodiments, the composition is suitable for systemic
administration, oral administration, intranasal administration or
parenteral administration.
[0023] In yet other aspects the present invention provides methods
for eliciting or enhancing an immune response in a mammal against
Neisseria, comprising administering to the mammal in an amount
effective to elicit or enhance the response, a Neisseria Hsp70
polypeptide in combination with a pharmaceutically acceptable
carrier or diluent; methods for eliciting or enhancing an immune
response in a mammal to a polypeptide comprising administering to
the mammal a fusion protein containing sequences of the polypeptide
fused to the Neisseria Hsp70 polypeptide in combination with a
pharmaceutically acceptable carrier or diluent; and methods for
eliciting or enhancing an immune response in a mammal against a
target antigen comprising administering to the mammal the target
antigen joined to a Neisseria Hsp70 polypeptide in combination with
a pharmaceutically acceptable carrier or diluent.
[0024] In still another aspect, this invention provides PCR primers
and probes for detecting DNA encoding a Neisseria meningitidis
Hsp70 that includes at least about 15 contiguous bases from any one
of SEQ. ID NOS: 1-4, or to compliment thereof. In a related aspect,
the invention provides a method for diagnosing the presence of a
Neisseria meningitidis in a subject sample that includes the steps
of obtaining a DNA fraction from the subject sample; and performing
a PCR amplification of the DNA fraction using at least one PCR
primer that includes at least about 15 contiguous bases from any
one of SEQ. ID NOS: 1-4, or a compliment thereof.
[0025] The present invention also provides an isolated nucleic acid
molecule encoding a Aspergillus fumigatus Hsp60. In some
embodiments, the isolated nucleotide molecule is selected from the
group consisting of: (a) an isolated nucleic acid molecule
comprising the sequence of SEQ ID NO: 5 (FIG. 14); (b) an isolated
nucleic acid molecule comprising the sequence of SEQ ID NO: 5 from
nucleotides 300-2234; (c) an isolated nucleic acid molecule
comprising the sequence of SEQ ID NO: 5 from nucleotides 300-410,
nucleotides 514-655 and nucleotides 724-2234; (d) an isolated
nucleic acid molecule comprising the sequence of SEQ ID NO: 6 (FIG.
16); (e) an isolated nucleic acid molecule comprising the sequence
of SEQ ID NO: 7 (FIG. 18); (f) an isolated nucleic acid molecule
complementary to any one of the nucleotides of SEQ ID NOS: 5 to 7
set forth in (a) through (e), respectively.
[0026] In another aspect, the present invention provides an
isolated nucleic acid molecule which is a variant of, or is
substantially similar to, the Aspergillus Hsp60 nucleotide
molecules described above. In further aspects the present invention
provides an isolated nucleic acid molecule comprising a nucleotide
sequence that is identical to a segment of contiguous nucleotide
bases comprising at least 25% of SEQ ID NOS: 5 to 7 or a complement
thereof or an isolated nucleic acid molecule encoding Hsp60
comprising a nucleic acid sequence that encodes a polypeptide
comprising any one of SEQ ID NOS: 5, 6 or 7 or a variant Hsp60 that
is at least 95% homologous to a polypeptide according to any one of
SEQ ID NOS: 5, 6, or 7.
[0027] In one embodiment, the present invention provides an
isolated nucleic acid molecule according as described above, the
molecule encoding a polypeptide that is able to be selectively
bound by an antibody specific for a Aspergillus fumigatus
Hsp60.
[0028] In still another aspect in one aspect the present invention
provides an isolated nucleic acid molecule encoding at least 8
contiguous amino acids of a Aspergillus fumigatus Hsp60 polypeptide
selected from amino acid residues according to FIG. 14, wherein the
encoded Aspergillus fumigatus Hsp60 polypeptide is able to bind to
a major histocompatibility complex.
[0029] In still further aspects the present invention provides an
isolated Aspergillus fumigatus Hsp60 polypeptide.
[0030] In some embodiments, the isolated Hsp60 polypeptide
comprises the amino acid sequence of FIG. 14, or variants thereof,
preferably wherein the polypeptide is able to be selectively bound
by an antibody specific for a Aspergillus fumigatus Hsp60. In
further embodiments, the isolated Hsp60 polypeptide is fused to an
additional polypeptide to create a fusion protein.
[0031] In still yet further aspects the present invention provides
an isolated Hsp60 polypeptide comprising at least 8 contiguous
amino acids selected from amino acid residues according to FIG. 14,
wherein the Hsp60 polypeptide is capable of binding to a major
histocompatibility complex and eliciting or enhancing an immune
response to Aspergillus fumigatus in a human being.
[0032] In certain embodiments, the isolated Hsp60 polypeptide is
derived from proteolytic cleavage or chemical synthesis, or is an
expression product of a transformed host cell containing a nucleic
acid molecule encoding the Hsp60 or portion thereof. In certain
further embodiments, the isolated Hsp60 polypeptide comprises
greater than 95% homology to the Hsp60 polypeptide of FIG. 14, and
the isolated Hsp60 polypeptide is able to be selectively bound by
an antibody specific for a Aspergillus fumigatus Hsp60.
[0033] In still yet another aspect the present invention provides
an isolated polypeptide wherein the polypeptide is an expression
product of a transformed host cell containing at least one of the
nucleic acid molecules derived from the aforementioned Aspergillus
molecules.
[0034] In still yet further aspects the present invention provides
vectors comprising at least one of the aforementioned nucleic acid
molecules derived from Aspergillus. In certain embodiments, the
vector is an expression vector comprising a promoter in operative
linkage with the isolated nucleic acid molecule encoding the Hsp60
or portion thereof, preferably further comprising a selectable or
identifiable marker and/or wherein the promoter is a constitutive
or an inducible promoter. The present invention also provides host
cells containing such vectors. In certain embodiments, the host
cell is selected from the group consisting of a bacterial cell, a
mammalian cell, a yeast cell, a plant cell and an insect cell.
[0035] In still yet other aspects the present invention provides
compositions comprising an Aspergillus Hsp60 polypeptide in
combination with a pharmaceutically acceptable carrier or diluent.
In certain embodiments, the composition is suitable for systemic
administration, oral administration, intranasal administration or
parenteral administration.
[0036] In yet other aspects the present invention provides methods
for eliciting or enhancing an immune response in a mammal against
Aspergillus, comprising administering to the mammal in an amount
effective to elicit or enhance the response, an Aspergillus Hsp60
polypeptide in combination with a pharmaceutically acceptable
carrier or diluent; methods for eliciting or enhancing an immune
response in a mammal to a polypeptide comprising administering to
the mammal a fusion protein containing sequences of the polypeptide
fused to the Hsp60 polypeptide in combination with a
pharmaceutically acceptable carrier or diluent; and methods for
eliciting or enhancing an immune response in a mammal against a
target antigen comprising administering to the mammal the target
antigen joined to an Aspergillus Hsp60 polypeptide in combination
with a pharmaceutically acceptable carrier or diluent.
[0037] In still another aspect, this invention provides PCR primers
and probes for detecting DNA encoding a Aspergillus fumigatus Hsp60
that includes at least about 15 contiguous bases from any one of
SEQ. ID NOS: 5-7, or to compliment thereof. In a related aspect,
the invention provides a method for diagnosing the presence of a
Aspergillus fumigatus in a subject sample that includes the steps
of obtaining a DNA fraction from the subject sample; and performing
a PCR amplification of the DNA fraction using at least one PCR
primer that includes at least about 15 contiguous bases from any
one of SEQ. ID NOS: 5-7, or a compliment thereof.
[0038] The present invention further provides an isolated nucleic
acid molecule encoding a Candida glabrata Hsp60. In some
embodiments, the isolated nucleotide molecule is selected from the
group consisting of: (a) an isolated nucleic acid molecule
comprising the sequence of SEQ ID NO: 8 (FIG. 21); (b) an isolated
nucleic acid molecule comprising the sequence of SEQ ID NO: 8 from
nucleotides 258-1964; (c) an isolated nucleic acid molecule
comprising the sequence of SEQ ID NO: 9 (FIG. 23); (d) an isolated
nucleic acid molecule comprising the sequence of SEQ ID NO: 10
(FIG. 25); (e) an isolated nucleic acid molecule complementary to
any one of the nucleotides of SEQ ID NOS: 8 to 10 set forth in (a)
through (d), respectively.
[0039] In another aspect, the present invention provides an
isolated nucleic acid molecule which is a variant of, or is
substantially similar to, the Candida Hsp60 nucleotide molecules
described above. In further aspects the present invention provides
an isolated nucleic acid molecule comprising a nucleotide sequence
that is identical to a segment of contiguous nucleotide bases
comprising at least 25% of SEQ ID NOS: 8 to 10 or a complement
thereof or an isolated nucleic acid molecule encoding Hsp60
comprising a nucleic acid sequence that encodes a polypeptide
comprising any one of the polypeptides according to FIGS. 21, 23 or
25, or a variant Hsp60 that is at least 95% homologous to a
polypeptide according to any one of FIGS. 21, 23 or 25.
[0040] In one embodiment, the present invention provides an
isolated nucleic acid molecule according as described above, the
molecule encoding a polypeptide that is able to be selectively
bound by an antibody specific for a Candida glabrata Hsp60.
[0041] In still another aspect in one aspect the present invention
provides an isolated nucleic acid molecule encoding at least 8
contiguous amino acids of a Candida glabrata Hsp60 polypeptide
selected from amino acid residues according to FIG. 21, wherein the
encoded Candida glabrata Hsp60 polypeptide is able to bind to a
major histocompatibility complex.
[0042] In still further aspects the present invention provides an
isolated Candida glabrata Hsp60 polypeptide.
[0043] In some embodiments, the isolated Hsp60 polypeptide
comprises the amino acid sequence of FIG. 21, or variants thereof,
preferably wherein the polypeptide is able to be selectively bound
by an antibody specific for a Candida glabrata Hsp60. In further
embodiments, the isolated Hsp60 polypeptide is fused to an
additional polypeptide to create a fusion protein.
[0044] In still yet further aspects the present invention provides
an isolated Hsp60 polypeptide comprising at least 8 contiguous
amino acids selected from amino acid residues according to FIG. 21,
wherein the Hsp60 polypeptide is capable of binding to a major
histocompatibility complex and eliciting or enhancing an immune
response to Candida glabrata in a human being.
[0045] In certain embodiments, the isolated Hsp60 polypeptide is
derived from proteolytic cleavage or chemical synthesis, or is an
expression product of a transformed host cell containing a nucleic
acid molecule encoding the Hsp60 or portion thereof. In further
certain embodiments, the isolated Hsp60 polypeptide comprises
greater than 95% homology to the Hsp60 polypeptide of FIG. 21, and
the isolated Hsp60 polypeptide is able to be selectively bound by
an antibody specific for a Candida glabrata Hsp60.
[0046] In still yet another aspect the present invention provides
an isolated polypeptide wherein the polypeptide is an expression
product of a transformed host cell containing at least one of the
aforementioned nucleic acid molecules derived from Candida.
[0047] In still yet further aspects the present invention provides
vectors comprising at least one of the aforementioned nucleic acid
molecules derived from Candida. In certain embodiments, the vector
is an expression vector comprising a promoter in operative linkage
with the isolated nucleic acid molecule encoding the Hsp60 or
portion thereof, preferably further comprising a selectable or
identifiable marker and/or wherein the promoter is a constitutive
or an inducible promoter. The present invention also provides host
cells containing such vectors. In certain embodiments, the host
cell is selected from the group consisting of a bacterial cell, a
mammalian cell, a yeast cell, a plant cell and an insect cell.
[0048] In still yet other aspects the present invention provides
compositions comprising a Candida Hsp60 polypeptide in combination
with a pharmaceutically acceptable carrier or diluent. In certain
embodiments, the composition is suitable for systemic
administration, oral administration, intranasal administration or
parenteral administration.
[0049] In yet other aspects the present invention provides methods
for eliciting or enhancing an immune response in a mammal against
Candida, comprising administering to the mammal in an amount
effective to elicit or enhance the response, a Candida Hsp60
polypeptide in combination with a pharmaceutically acceptable
carrier or diluent; methods for eliciting or enhancing an immune
response in a mammal to a polypeptide comprising administering to
the mammal a fusion protein containing sequences of the polypeptide
fused to the Candida Hsp60 polypeptide in combination with a
pharmaceutically acceptable carrier or diluent; and methods for
eliciting or administering to the mammal the target antigen joined
to a Candida Hsp60 polypeptide in combination with a
pharmaceutically acceptable carrier or diluent.
[0050] In still another aspect, this invention provides PCR primers
and probes for detecting DNA encoding a Candida glabrata Hsp60 that
includes at least about 15 contiguous bases from any one of SEQ. ID
NOS: 8-10, or to compliment thereof. In a related aspect, the
invention provides a method for diagnosing the presence of Candida
glabrata in a subject sample that includes the steps of obtaining a
DNA fraction from the subject sample; and performing a PCR
amplification of the DNA fraction using at least one PCR primer
that includes at least about 15 contiguous bases from any one of
SEQ. ID NOS: 8-10, or a compliment thereof.
[0051] These and other aspects of the present invention will become
evident upon reference to the present specification and the
attached drawings. In addition, various references are set forth
herein that describe in more detail certain procedures or
compositions (e.g., plasmids, etc.); all such references are
incorporated herein in their entirety by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1 illustrates the strategy employed to obtain the
nucleotide sequence of an internal fragment of the Neisseria
meningitidis Hsp70 gene.
[0053] FIG. 2 depicts the nucleotide and amino acid sequences of an
internal fragment of the Neisseria meningitidis Hsp70 gene.
[0054] FIG. 3 illustrates the strategy used to obtain the
nucleotide and amino acid sequences of the Neisseria meningitidis
Hsp 70 gene.
[0055] FIG. 4 depicts first nucleotide and amino acid sequences of
Neisseria meningitidis Hsp70 gene. SEQ.ID.NO. 1.
[0056] FIG. 5 illustrates strategy employed to obtain second
sequences of Neisseria meningitis Hsp70 gene.
[0057] FIG. 6 depicts second nucleotide and amino acid sequences of
Neisseria meningitidis Hsp70 gene. SEQ.ID.NO. 2.
[0058] FIG. 7 illustrates the strategy used to obtain nucleic acid
and amino acid sequences of the Neisseria meningitidis Hsp70 genes
cloned into pET24A+ and pET28A+.
[0059] FIG. 8 depicts the nucleotide and amino acid sequences of
Neisseria meningitidis Hsp70 gene cloned into pET24A+. SEQ.ID.NO.
3.
[0060] FIG. 9 depicts the nucleotide and amino acid sequences of
Neisseria meningitidis Hsp70 gene cloned into pET28A+. SEQ.ID.NO.
4.
[0061] FIG. 10 shows a stained SDS-PAGE gel illustrating expression
of recombinant Neisseria meningitidis Hsp70.
[0062] FIG. 11 shows a stained SDS-PAGE gel illustrating
purification of recombinant Neisseria meningitidis Hsp70.
[0063] FIG. 12 shows an EtBr-stained gel illustrating selective
amplification of Neisseria meningitidis Hsp70 and Streptococcal
Hsp70 gene sequences.
[0064] FIG. 13 illustrates the strategy employed to obtain second
nucleic acid and amino acid sequences of the Aspergillus fumigatus
Hsp60 gene.
[0065] FIG. 14 depicts the nucleotide and amino acid sequences of
Aspergillus fumigatus Hsp60 gene. SEQ. ID. NO. 5.
[0066] FIG. 15 shows the map of expression plasmid pETAF60.
[0067] FIG. 16 depicts the nucleotide and amino acid sequences of
Aspergillus fumigatus Hsp60 gene in plasmid pETAF60. SEQ.ID.NO.
6.
[0068] FIG. 17 shows the map of expression plasmid pETAF60H.
[0069] FIG. 18 depicts the nucleotide and amino acid sequences of
Aspergillus fumigatus Hsp60 gene in plasmid pETAF60H. SEQ.ID.NO.
7.
[0070] FIG. 19 shows a stained SDS-PAGE gel illustrating expression
of recombinant Aspergillus fumigatus Hsp60.
[0071] FIG. 20 illustrates the strategy employed to obtain nucleic
acid and amino acid sequences of the Candida glabrata Hsp60
gene.
[0072] FIG. 21 depicts the nucleotide and amino acid sequences of
Candida glabrata Hsp60 gene. SEQ.ID.NO. 8.
[0073] FIG. 22 shows the map of expression plasmid pETCG60A.
[0074] FIG. 23 depicts the nucleotide and amino acid sequences of
Candida glabrata Hsp60 gene in plasmid pETCG60A. SEQ.ID.NO. 9.
[0075] FIG. 24 shows the map of expression plasmid pETCG60AH.
[0076] FIG. 25 depicts the nucleotide and amino acid sequences of
Candida glabrata Hsp60 gene in plasmid pETCGA60H. SEQ.ID.NO.
10.
[0077] FIG. 26 shows a stained SDS-PAGE gel illustrating expression
of recombinant Candida glabrata Hsp60.
[0078] FIG. 27 shows a stained SDS-PAGE gel illustrating
purification of recombinant Candida glabrata Hsp60.
DETAILED DESCRIPTION OF THE INVENTION
[0079] The present invention provides methods and compositions
comprising isolated nucleic acid molecules and polypeptides
specific to Neisseria menigitidis, Aspergillus fumigatus and
Candida glabrata, as well as vector constructs, antibodies and
other materials related to isolated nucleic acid molecules and
polypeptides. Such compositions and methods are useful for the
diagnosis of Neisserial, Aspergillal and Candidal infection and for
generating (eliciting or enhancing) an immune response to these
organisms.
[0080] Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of certain terms
to be used hereafter.
[0081] A "stress gene," also known as "heat shock gene," is a gene
that is activated or otherwise detectably upregulated due to the
contact or exposure of an organism (containing the gene) to a
stressor, such as heat shock or glucose deprivation or glucose
addition. A given "stress gene" also includes homologous genes
within known stress gene families, such as certain genes within the
Hsp60, Hsp70 and Hsp90 stress gene families, even though such
homologous genes are not themselves induced by a stressor.
[0082] A "stress protein," also known as a "heat shock protein,"
("Hsp") is a protein that is encoded by a stress gene, and is
therefore typically produced in significantly greater amounts upon
the contact or exposure to the stressor of the organism. Each of
the terms stress gene and stress protein as used in the present
specification are inclusive of the other, unless the context
indicates otherwise. Neisserial, Aspergillal and Candidal Hsps, as
well as Hsps from other organisms, appear to participate in
important cellular processes such as protein synthesis and assembly
and disassembly of protein complexes.
[0083] As used herein, "polypeptide" refers to full length proteins
and fragments thereof.
[0084] As used herein, "peptide" refers to a fragment of the whole
protein, whether chemically or biologically produced.
[0085] As used herein, "immunogenic" refers to an antigen or
composition that elicits an immune response.
[0086] An "isolated nucleic acid molecule" refers to a
polynucleotide molecule, in the form of a separate fragment or as a
component of a larger nucleic acid construct, that has been
separated from its source cell (including the chromosome it
normally resides in) at least once in a substantially pure form.
Nucleic acid molecules can be comprised of a wide variety of
nucleotides and molecules well known in the art, including DNA,
RNA, nucleic acid analogues, or any combination of these.
[0087] As used herein, "vector" refers to a polynucleotide assembly
capable of directing expression and/or replication of the nucleic
acid sequence of interest. Such assembly can, if desired, be
included as a part of other components, such as a protein, lipid or
lipoprotein coat, for delivery of the vector or for other
purposes.
[0088] An "expression vector" refers to polynucleotide vector
having at least a promoter sequence operably linked to the nucleic
acid sequence of interest.
[0089] As used herein, a "promoter" refers to a nucleotide sequence
that contains elements that direct the transcription of an operably
linked nucleic acid sequence. At minimum, a promoter contains an
RNA polymerase binding site. Promoter regions can also contain
enhancer elements which by definition enhance transcription.
[0090] A. Hsp Genes and Polypeptides from Neisseria meningitidis,
Aspergillus fumigatus and Candida glabrata
[0091] As used herein, "Hsp70" refers to heat shock genes from a
Hsp70 family of genes that encode heat shock proteins of
approximately 70 kDa, and the heat shock gene products encoded
thereby. The nucleotide and amino acid sequences of Hsp70 genes and
gene products from Neisseria meningitidis are set forth in FIGS. 4,
6, 8 and 9 (SEQ ID NOS: 1-4; such sequences also include the PCR
primers used to isolate the Hsp70 genes). As used herein, Hsp60
refers to heat shock genes from the Hsp60 family of genes that
encode heat shock proteins of approximately 60 kDa; and the heat
shock gene products encoded thereby. The nucleotide and amino acid
sequences of Hsp60 genes and gene products from Aspergillus
fumigatus and Candida glabrata are set forth in FIGS. 14, 16, 18,
21, 23 and 25 (SEQ ID NOS: 5-10; such sequences also include the
PCR primers used to isolate the Hsp60 genes).
[0092] Within the context of this invention it should be understood
that Hsp70 and Hsp60 include wild-type/native protein sequences, as
well as other variants (including alleles) and fragments of the
native protein sequences. Briefly, such variants may result from
natural polymorphisms or be synthesized by recombinant methodology
or chemical synthesis, and differ from wild-type proteins by one or
more amino acid substitutions, insertions, deletions, or the like.
Further, in the region of homology to the native sequence, a
variant should preferably have at least 95% amino acid sequence
homology, and within certain embodiments, greater than 97% or 98%
homology. As used herein, amino acid "homology" is determined by a
computer algorithm incorporated in a protein database search
program commonly used in the art, and more particularly, as
incorporated in the programs BLAST.TM. (Altschul et al., Nucleic
Acids Res. (25) 3389-3402, 1997) or DNA STAR MEGALIGN.TM. which
return similar results in homology calculations. As will be
appreciated by those of ordinary skill in the art, a nucleotide
sequence encoding an Hsp or a variant may differ from the native
sequences presented herein due to codon degeneracies, nucleotide
polymorphisms, or nucleotide substitutions, deletions or
insertions.
[0093] The aforementioned sequences are useful for generating PCR
primers and probes for the detection of Neisseria meningitidis,
Aspergillus fumigatus or Candida glabrata. Thus, one aspect of this
invention includes PCR primers and probes for detecting DNA
encoding the Hsps disclosed herein. Useful PCR primers typically
include at least about 15 contiguous bases from the nucleic acid
sequences provided herein, or compliments thereof More
particularly, PCR primers and probes for detection of Neisseria
meningitidis include at least about 15 contiguous bases from any
one SEQ. ID NOS: 1-4 or compliments thereof; PCR primers and probes
for detection of Aspergillus fumigatus include at least about 15
contiguous bases from any one SEQ. ID NOS: 5-7 or compliments
thereof; and PCR primers and probes for detection of Candida
glabrata include at least about 15 contiguous bases from any one
SEQ. ID NOS: 8-10 or compliments thereof. In certain embodiments,
PCR primers derived from these sequences can be used in a
diagnostic method to detect the presence of a specific
microorganism in a subject sample by amplifying DNA isolated from
the subject sample to detect the Hsp genes present in any one of
Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata
as opposed to other microorganisms. Thus for example, primer pairs
comprising 5' CTGCCGTACATCACCATGG 3' with 5'
GGCTTCTTGTACTTTCGGC-3'; were able to specifically amplify DNA
isolated from Neisseria meningitidis, but not other microorganisms
known to contain Hsps. Similarly, primer pairs derived from an
Hsp70 gene isolated from Streptococcus (5' TGACCTTGTTGAACGTAC-3'
with 5' ACTTCATCAGGGTTTAC-3') were able to amplify DNA isolated
from Streptococcus but not Neisseria (See Example 5).
[0094] An "isolated nucleic acid molecule encoding Neisseria
meningitidis Hsp70, Aspergillus fumigatus Hsp60 or Candida glabrata
Hsp60" refers to nucleic acid sequences that are capable of
encoding Hsp70 or Hsp60 polypeptides of these organisms. While
several embodiments of such molecules are depicted in SEQ ID NOS:
1-10, it should be understood that within the context of the
present invention, reference to one or more of these molecules
includes variants that are naturally occurring and/or synthetic
sequences which are substantially similar to the sequences provided
herein and, where appropriate, the protein (including peptides and
polypeptides) that are encoded by these sequences and their
variants. As used herein, the nucleotide sequence is deemed to be
"substantially similar" if: (a) the nucleotide sequence is derived
from the coding region of a native gene of Neisseria menigitidis,
Aspergillus fumigatus or Candida glabrata and encodes a peptide or
polypeptide that binds to an antibody or HLA molecule that
specifically binds to a polypeptide described herein (including,
for example, portions of the sequence or allelic variations of the
sequences discussed above); or (b) the nucleotide sequences are
degenerate (i.e., sequences which encode the same amino acid using
a different codon sequence) as a result of the genetic code to the
nucleotide sequences defined in (a); or (c) the nucleotide sequence
is at least 95% identical to a nucleotide sequence provide herein,
or (d) is a complement of any of the sequences described in (a-c).
As used herein, "high stringency" are conditions for hybridization
of nucleic acids as described in units 6.3 and 6.4 by Ausubel et
al., Current Protocols in Molecular Biology, vol. 1. John Wiley
& Sons (1998).
[0095] One aspect of the present invention is the use of Neisseria
meningitidis Hsp70, Aspergillus fumigatus Hsp60 or Canadida
glabrata Hsp60 nucleotide sequences to produce recombinant proteins
for immunizing an animal. Therefore, the use of any length of
nucleic acid disclosed by the present invention (preferably 24
nucleotides or longer) which encodes a polypeptide or fragment
thereof that is capable of binding to the major histocompatibility
complex and eliciting or enhancing an immunogenic response is
contemplated by this invention. Immunogenic response can be readily
tested by known methods such as challenging a mouse or rabbit with
the antigen of interest and thereafter collecting plasma and
determining if antibodies of interest are present. Other assays
particularly useful for the detection of T-cell responses include
proliferation assays, T-cell cytotoxicity assays and assays for
delayed hypersensitivity. In determining whether an antibody
specific for the antigen of interest was produced by the animal,
many diagnostic tools are available, for example, testing binding
of labeled antigen to plasma derived antibodies, or using
Enzyme-linked immunoassays with tag attached to the antigen of
interest.
[0096] The Neisseria meningitidis Hsp70, Aspergillus fumigatus
Hsp60 and Canadida glabrata Hsp60 genes of this invention can be
obtained using a variety of methods. For example, a nucleic acid
molecule can be obtained from a cDNA or genomic expression library
by screening with an antibody or antibodies reactive to one or more
of these Hsps (see, e.g, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, 1989; Ausubel et al.,
Current Protocols in Molecular Biology, Greene Publishing, 1987).
Further, random-primed PCR can be employed (see, e.g., Methods in
Enzymol. 254:275, 1995). In one such method, one of the primers is
a poly deoxy-thymine and the other is a degenerate primer based on
the amino acid sequence or nucleotide sequence of related Hsps.
[0097] Other methods can also be used to obtain a nucleic acid
molecule that encodes Neisseria meningitidis Hsp70, Aspergillus
fumigatus Hsp60 or Canadida glabrata Hsp60. For example, a nucleic
acid molecule can be obtained by using the sequence information
provided herein to synthesize a probe which can be labeled, such as
with a radioactive label, enzymatic label, protein label,
fluorescent label, or the like, and hybridized to a genomic library
or a cDNA library constructed in a phage, plasmid, phagemid, viral,
or other vector (see, e.g., Sambrook et al. (supra); Ausubel et al.
(supra)). DNA representing RNA or genomic nucleic acid sequence can
also be obtained by amplification using sets of primers
complementary to 5' and 3' sequences of the cDNA sequence, such as
presented in the Examples. For ease of cloning, restriction sites
can also be incorporated into the primers.
[0098] Variants (including alleles) of the Hsp70 and Hsp60 genes
provided herein can be readily isolated from natural sources
containing such variants (e.g., polymorphisms, mutants), or can be
synthesized or constructed using recombinant DNA and mutagenesis
techniques known in the art. Many methods have been developed for
generating mutants (see generally Sambrook et al. (supra); Ausubel
et al. (supra)). Briefly, preferred methods for generating
nucleotide substitutions utilize an oligonucleotide that spans the
base or bases to be mutated and contains the mutated base or bases.
The oligonucleotide is hybridized to complementary single stranded
nucleic acid and second strand synthesis is primed from the
oligonucleotide. The double-stranded nucleic acid is prepared for
transformation into host cells, such as E. coli, other prokaryotes,
yeast or other eukaryotes. Standard screening and vector growth
protocols are used to identify mutant sequences and obtain high
yields.
[0099] Similarly, deletions and/or insertions of the Hsp70 or the
Hsp60 gene can be constructed by any of a variety of known methods.
For example, the gene can be digested with restriction enzymes and
religated such that sequence is deleted or religated with
additional sequence such that an insertion or large substitution is
made. Other means of generating variant sequences, known in the
art, can be employed, for examples see Sambrook et al. (supra) and
Ausubel et al. (supra). Moreover, verification of variant sequences
is typically accomplished by restriction enzyme mapping, sequence
analysis or hybridization. Variants which encode a polypeptide that
elicits an immunogenic response specific for Neisseria
meningitidis, Aspergillus fumigatus or Candida glabrata are useful
in the context of this invention.
[0100] As noted above, the present invention also provides isolated
polypeptides. Within the context of the present invention, unless
otherwise clear from the context, such polypeptides are understood
to include the whole, or portions/fragments, of a gene product
derived from one or more of the Hsp70 and Hsp60 genes of the
invention or variants thereof as discussed above. In one aspect of
the present invention, the protein is encoded by a portion of a
native gene or is encoded by a variant of a native gene and the
protein or fragment thereof elicits or enhances an immune response
specific for Neisseria meningitidis, Aspergillus fumigatus or
Candida glabrata.
[0101] A "purified" Hsp70 or Hsp60 stress protein of the present
invention is a heat shock protein of the Hsp70 or Hsp60 family from
Neisseria meningitidis (Hsp70), Aspergillus fumigatus (Hsp60) or
Candida glabrata (Hsp60) that has been purified from its producing
cell. For example, the Hsp70 and Hsp60 polypeptides of the present
invention can be purified by a variety of standard methods with or
without a detergent purification step. For example, Hsp70 or Hsp60
of the present invention can be isolated by, among other methods,
culturing suitable host and vector systems to produce recombinant
Hsps (discussed further herein). Then, supernatants from such cell
lines, or Hsp inclusions, or whole cells where the Hsp is not
excreted into the supernatant, can be treated by a variety of
purification procedures. For example, the Hsp-containing
composition can be applied to a suitable purification matrix such
as an anti-Hsp60 antibody bound to a suitable support.
Alternatively, anion or cation exchange resins, gel filtration or
affinity, hydrophobic or reverse phase chromatography may be
employed in order to purify the protein. The Hsp polypeptide can
also be concentrated using commercially available protein
concentration filters, such as an Amicon or Millipore Pellicon
ultrafiltration unit, or by vacuum dialysis. In another
alternative, when the polypeptide is secreted the supernatant
medium containing the polypeptide can first be concentrated using
one of the above mentioned protein concentration filters, followed
by application of the concentrate to a suitable purification matrix
such as those described above.
[0102] In one embodiment, the isolated Hsp70 and Hsp60s of the
present invention are produced in a recombinant form, utilizing
genetic manipulation techniques that are well known in the art. For
example, a Hsp of the present invention can be expressed as a
histidine-tagged molecule, permitting purification on a
nickel-chelating matrix. Alternatively, other tags may be used,
including FLAG and GST. The associated tag can then be removed in
the last step of purification, for example, for certain vectors,
His-tagged proteins may be incubated with thrombin, resulting in
cleavage of a recognition sequence between the tag and the Hsp
polypeptide (e.g., pET vectors from Invitrogen). Following
purification of an Hsp of the invention from a gram-negative
bacterial host, whether tagged or not, it will be necessary to
reduce the level of endotoxin in the Hsp preparation.
[0103] B. Vectors, Host Cells, and Expression of Hsps from
Neisseria meningitis, Aspergillus fumigatus and Candida
glabrata
[0104] It is well known in the art that certain vectors (e.g., pUC)
can be used for producing multiple copies of a nucleotide molecule
of interest as well as being useful for genetic manipulation
techniques (e.g., site-directed mutagenesis). See Sambrook (supra).
Expression vectors are particularly suited to the practice of this
invention. An expression vector includes transcriptional
promoter/enhancer elements operably linked to the Neisserial,
Aspergillal or Candidal Hsp nucleic acid molecule and may typically
contain a selectable or otherwise identifiable marker gene. The
expression vector may be composed of either deoxyribonucleic acids
("DNA"), ribonucleic acids ("RNA"), or a combination of the two
(e.g., a DNA-RNA chimera). Optionally, the expression vector may
include a polyadenylation sequence or one or more restriction
sites. Additionally, depending on the host cell chosen and the
expression vector employed, other genetic elements such as an
origin of replication, additional nucleic acid restriction sites,
enhancers, sequences conferring inducibility of transcription, and
genes encoding proteins suitable for use as selectable or
identifiable markers, may also be incorporated into the expression
vectors described herein.
[0105] The manipulation and expression of Neisserial, Aspergillal
or Candidal Hsp genes can be accomplished by culturing host cells
containing an expression vector capable of expressing the Hsp
genes. Such vectors or vector constructs include either synthetic
or cDNA-derived nucleic acid molecules or genomic DNA fragments
encoding the Hsp polypeptides, which are operably linked to
suitable transcriptional or translational regulatory elements.
Suitable regulatory elements within the expression vector can be
derived from a variety of sources, including bacterial, fungal,
viral, mammalian, insect, or plant genes. Selection of appropriate
regulatory elements is dependent on the host cell chosen, and can
be readily accomplished by one of ordinary skill in the art in
light of the present specification. Examples of regulatory elements
include a transcriptional promoter and enhancer or RNA polymerase
binding sequence, a transcriptional terminator, and a ribosomal
binding sequence, including a translation initiation signal.
[0106] Nucleic acid molecules that encode any of the Neisserial,
Aspergillal or Candidal Hsp polypeptides described above can be
expressed by a wide variety of prokaryotic and eukaryotic host
cells, including bacterial, mammalian, yeast or other fungi, viral,
insect, and plant cells. The selection of a host cell may also
assist the production of post-translationally modified Hsps (e.g.
modified by glycosylation, prenylation, acetylation or other
processing event), depending upon the desires of the user. Methods
for transforming or transfecting such cells to express nucleic
acids are well known in the art (see, e.g. Itakura et al., U.S.
Pat. No. 4,704,362; Hinnen et al., PNAS USA 75:1929-1933. 1978;
Murray et al., U.S. Pat. No. 4,801,542; Upshall et al., U.S. Pat.
No. 4,935.349; Hagen et al. U.S. Pat. No. 4,784,950; Axel et al.,
U.S. Pat. No. 4,399,216; Goeddel et al., U.S. Pat. No. 4,766,075;
and Sambrook et al., Molecular Cloning. A Laboratory Manual,
2.sup.nd edition, Cold Spring Harbor Laboratory Press, 1989; for
plant cells see Czako and Marton, Plant Physiol. 104:1067-1071,
1994; Paszkowski et al., Biotech. 24:387-392, 1992).
[0107] Bacterial host cells suitable for carrying out the present
invention include E. coli, such as E. coli DH5.alpha. (Stratagene,
La Jolla, Calif.) or BL21 (DE3) (Novagen, Madison, Wis.), M.
leprae, M. tuberculosis, M. bovis, B. subtilis, Salmonella
typhimurium, and various species within the genera Pseudomonas,
Streptomyces, Streptococcus, and Staphylococcus, as well as many
other bacterial species well known to one of ordinary skill in the
art.
[0108] Bacterial expression vectors preferably comprise a promoter,
which functions in the host cell, one or more selectable phenotypic
markers, and a bacterial origin of replication. Representative
promoters include the .beta.-lactamase (penicillinase) and lactose
promoter system (see Chang et al., Nature 275:615, 1978), the T7
RNA polymerase promoter (Studier et al., Meth. Enzymol. 185:60-89,
1990), the lambda promoter (Elvin et al., Gene 87:123-126, 1990),
the trp promoter (Nichols and Yanofsky, Meth. in Enzymology
101:155, 1983) and the tac promoter (Russell et al., Gene 20: 231,
1982). Representative selectable markers include various antibiotic
resistance markers such as the kanamycin or ampicillin resistance
genes. Many plasmids suitable for transforming host cells are well
known in the art, including among others, pBR322 (see Bolivar et
al., Gene 2:95, 1977), the pUC plasmids pUC18, pUC19, pUC118,
pUC119 (see Messing, Meth. in Enzymology 101:20-77, 1983; Vieira
and Messing, Gene 19:259-268, 1982), and pNH8A, pNH16a, pNH18a, and
Bluescript M13 (Stratagene, La Jolla, Calif.).
[0109] Fungal host cells suitable for carrying out the present
invention include, among others, Saccharomyces pombe, Saccharomyces
cerevisiae, the genera Pichia or Kluyveromyces and various species
of the genus Aspergillus (McKnight et al., U.S. Pat. No.
4,935,349). Suitable expression vectors for yeast and fungi
include, among others, YCp50 (ATCC No. 37419) for yeast, and the
amdS cloning vector pV3 (Tumbull, Bio/Technology 7:169, 1989), YRp7
(Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039, 1978),
YEp13 (Broach et al., Gene 8:121-133, 1979), pJDB249 and pJDB219
(Beggs, Nature 275:104-108, 1978) and derivatives thereof.
[0110] Preferred promoters for use in yeast include promoters from
yeast glycolytic genes (Hitzeman et al., J. Biol. Chem.
255:12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet.
1:419-434, 1982) or alcohol dehydrogenase genes (Young et al., in
Genetic Engineering of Microorganisms for Chemicals, Hollaender et
al. (eds.), p. 355, Plenum, New York, 1982; Ammerer, Meth. Enzymol.
101:192-201, 1983). Examples of useful promoters for fungi vectors
include those derived from Aspergillus nidulans glycolytic genes,
such as the adh3 promoter (McKnight et al., EMBO J. 4:2093-2099,
1985). The expression units may also include a transcriptional
terminator. An example of a suitable terminator is the adh3
terminator (McKnight et al., ibid., 1985).
[0111] As with bacterial vectors, the yeast vectors will generally
include a selectable marker, which may be one of any number of
genes that exhibit a dominant phenotype for which a phenotypic
assay exists to enable transformants to be selected. Preferred
selectable markers include those that complement host cell
auxotrophy, provide antibiotic resistance or enable a cell to
utilize specific carbon sources, and include leu2 (Broach et al.,
ibid.), ura3 (Botstein et al., Gene 8:17, 1979), or his3 (Struhl et
al., ibid.). Another suitable selectable marker is the cat gene,
which confers chloramphenicol resistance on yeast cells.
[0112] Techniques for transforming fungi are well known in the
literature, and have been described, for instance, by Beggs
(ibid.), Hinnen et al. (Proc. Natl. Acad. Sci. USA 75:1929-1933,
1978), Yelton et al. (Proc. Natl. Acad. Sci. USA 81:1740-1747,
1984), and Russell (Nature 301:167-169, 1983). The genotype of the
host cell may contain a genetic defect that is complemented by the
selectable marker present on the expression vector. Choice of a
particular host and selectable marker is well within the level of
ordinary skill in the art in light of the present
specification.
[0113] Protocols for the transformation of yeast are also well
known to those of ordinary skill in the art. For example,
transformation may be readily accomplished either by preparation of
spheroplasts of yeast with DNA (see Hinnen et al., PNAS USA
75:1929, 1978) or by treatment with alkaline salts such as LiCl
(see Itoh et al., J. Bacteriology 153:163, 1983). Transformation of
fungi may also be carried out using polyethylene glycol as
described by Cullen et al. (Bio/Technology 5:369, 1987).
[0114] Viral vectors include expression vectors that comprise a
promoter which directs the expression of an isolated nucleic acid
molecule encoding a Hsp according to this invention. A wide variety
of promoters may be utilized within the context of the present
invention, including for example, promoters such as MoMLV LTR, RSV
LTR, Friend MuLV LTR, adenoviral promoter (Ohno et al., Science
265: 781-784, 1994), neomycin phosphotransferase promoter/enhancer,
late parvovirus promoter (Koering et al., Hum. Gene Therap.
5:457-463, 1994), Herpes TK promoter, SV40 promoter,
metallothionein IIa gene enhancer/promoter, cytomegalovirus
immediate early promoter, and the cytomegalovirus immediate late
promoter. The promoter may also be a tissue-specific promoter (see
e.g., WO 91/02805; EP 0,415,731; and WO 90/07936). In addition to
the above-noted promoters, other viral-specific promoters (e.g.,
retroviral promoters (including those noted above, as well as
others such as HIV promoters), hepatitis, herpes (e.g., EBV), and
bacterial, fungal or parasitic-specific (e.g., malarial-specific)
promoters may be utilized in order to target a specific cell or
tissue which is infected with a virus, bacteria, fungus or
parasite.
[0115] Thus, Neisserial Hsp70, Aspergillal Hsp60 or Candidal Hsp60
polypeptides of the present invention may be expressed from a
variety of viral vectors, including for example, herpes viral
vectors (e.g., U.S. Pat. No. 5,288,641), adenoviral vectors (e.g.,
WO 94/26914, WO 93/9191; Kolls et al., PNAS 91(1):215-219, 1994;
Kass-Eisler et al., PNAS 90(24):11498-502, 1993; Guzman et al.,
Circulation 88(6):2838-48, 1993; Guzman et al., Cir. Res.
73(6):1202-1207, 1993; Zabner et al., Cell 75(2):207-216, 1993; Li
et al., Hum Gene Ther. 4(4):403-409, 1993; Caillaud et al., Eur. J.
Neurosci. 5(10):1287-1291, 1993; Vincent et al., Nat. Genet.
5(2):130-134, 1993; Jaffe et al., Nat. Genet. 1(5):372-378, 1992;
and Levrero et al., Gene 101(2):195-202, 1991),
adenovirus-associated viral vectors (Flotte et al., PNAS
90(22):10613-10617, 1993), baculovirus vectors, parvovirus vectors
(Koering et al., Hum. Gene Therap. 5:457-463, 1994), pox virus
vectors (Panicali and Paoletti, PNAS 79:4927-4931, 1982; and Ozaki
et al., Biochem. Biophys. Res. Comm. 193(2):653-660, 1993), and
retroviruses (e.g., EP 0,415,731; WO 90/07936; WO 91/0285, WO
94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO
93/11230; WO 93/10218. Within various embodiments, either the viral
vector itself or a viral particle which contains the viral vector
may be utilized in the methods and compositions described
below.
[0116] Mammalian cells suitable for carrying out the present
invention include, among others: PC12 (ATCC No. CRL1721), N1E-115
neuroblastoma, SK-N-BE(2)C neuroblastoma, SHSY5 adrenergic
neuroblastoma, NS20Y and NG108-15 murine cholinergic cell lines, or
rat F2 dorsal root ganglion line, COS (e.g., ATCC No. CRL 1650 or
1651), BHK (e.g., ATCC No. CRL 6281; BHK 570 cell line (deposited
with the American Type Culture Collection under accession number
CRL 10314), CHO (ATCC No. CCL61), HeLa (e.g., ATCC No. CCL 2), 293
(ATCC No. 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and
NS-1 cells. Other mammalian cell lines may be used within the
present invention, including Rat Hep I (ATCC No. CRL 1600), Rat Hep
II (ATCC No. CRL 1548), TCMK (ATCC No. CCL 139), Human lung (ATCC
No. CCL 75.1), Human hepatoma (ATCC No. HTB-52), Hep G2 (ATCC No.
HB 8065), Mouse liver (ATCC No. CCL 29.1), NCTC 1469 (ATCC No. CCL
9.1), SP2/0-Ag14 (ATCC No. 1581), HIT-T15 (ATCC No. CRL 1777), and
RINm 5AHT2B (Orskov and Nielson, FEBS 229(1):175-178, 1988).
[0117] Mammalian expression vectors for use in carrying out the
present invention include a promoter capable of directing the
transcription of a cloned gene or cDNA. Preferred promoters include
viral promoters and cellular promoters. Example viral promoters
include the cytomegalovirus immediate early promoter (Boshart et
al., Cell 41:521-530, 1985), cytomegalovirus immediate late
promoter, SV40 promoter (Subramani et al., Mol. Cell. Biol.
1:854-864, 1981), MMTV LTR, RSV LTR, and adenovirus Ela. Example
cellular promoters include the mouse metallothionein-1 promoter
(Palmiter et al., U.S. Pat. No. 4,579,821), actin promoters, a
mouse V.sub.H promoter (Bergman et al., Proc. Natl. Acad. Sci. USA
81:7041-7045, 1983; Grant et al., Nucl. Acids Res. 15:5496, 1987)
and a mouse V.sub.H promoter (Loh et al., Cell 33:85-93, 1983). The
choice of promoter will depend, at least in part, upon the level of
expression desired or the recipient cell line to be
transfected.
[0118] Such expression vectors can also contain a set of RNA splice
sites located downstream from the promoter and upstream from the
DNA sequence encoding the peptide or protein of interest. Preferred
RNA splice sites may be obtained from adenovirus and/or
immunoglobulin genes. Also contained in the expression vectors is a
polyadenylation signal located downstream of the coding sequence of
interest. Suitable polyadenylation signals include the early or
late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.),
the polyadenylation signal from the Adenovirus 5 E1B region and the
human growth hormone gene terminator (DeNoto et al., Nuc. Acids
Res. 9:3719-3730, 1981). The expression vectors may include a
noncoding viral leader sequence, such as the Adenovirus 2
tripartite leader, located between the promoter and the RNA splice
sites. Preferred vectors may also include enhancer sequences, such
as the SV40 enhancer. Expression vectors may also include sequences
encoding the adenovirus VA RNAs. Suitable expression vectors can be
obtained from commercial sources (e.g., Stratagene, La Jolla,
Calif.).
[0119] Vector constructs comprising cloned DNA sequences can be
introduced into cultured mammalian cells by, for example, calcium
phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978;
Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and
Van der Eb, Virology 52:456, 1973), electroporation (Neumann et
al., EMBO J. 1:841-845, 1982), or DEAE-dextran mediated
transfection (Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., NY, 1987). See generally
Sambrook et al. (supra). To identify cells that have stably
integrated the cloned DNA, a selectable marker is generally
introduced into the cells along with the gene or cDNA of interest.
Preferred selectable markers for use in cultured mammalian cells
include genes that confer resistance to drugs, such as neomycin,
hygromycin, and methotrexate. The selectable marker may be an
amplifiable selectable marker. Preferred amplifiable selectable
markers are the DHFR gene and the neomycin resistance gene.
Selectable markers are reviewed by Thilly (Mammalian Cell
Technology, Butterworth Publishers, Stoneham, Mass.).
[0120] Mammalian cells containing a suitable vector are allowed to
grow for a period of time, typically 1-2 days, to begin expressing
the DNA sequence(s) of interest. Drug selection is then applied to
select for growth of cells that are expressing the selectable
marker in a stable fashion. For cells that have been transfected
with an amplifiable, selectable marker the drug concentration may
be increased in a stepwise manner to select for increased copy
number of the cloned sequences, thereby increasing expression
levels. Cells expressing the introduced sequences are selected and
screened for production of the protein of interest in the desired
form or at the desired level. Cells that satisfy these criteria can
then be cloned and scaled up for production.
[0121] Numerous insect host cells known in the art can also be
useful within the present invention, in light of the subject
specification. For example, the use of baculoviruses as vectors for
expressing heterologous DNA sequences in insect cells has been
reviewed by Atkinson et al. (Pestic. Sci. 28:215-224,1990).
[0122] Numerous plant host cells known in the art can also be
useful within the present invention, in light of the subject
specification. For example, the use of Agrobacterium rhizogenes as
vectors for expressing genes in plant cells has been reviewed by
Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.
[0123] Upon expression of the Neisserial Hsp70, Aspergillal Hsp60
or Candidal Hsp60 polypeptides or fragments thereof in the host
cells, the polypeptide or peptide may be released and/or isolated
from the host cell utilizing methods such as those discussed
previously herein.
[0124] As noted above, depending on the host cell in which one
desires to express an Neisserial, Aspergillal or Candidal Hsp, the
gene encoding the protein is introduced into an expression vector
comprising a promoter that is active in the host cell. Other
components of the expression unit such as transcribed but not
translated sequences at the ends of the coding region may also be
selected according to the particular host utilized. In some cases,
it may be necessary to introduce artificially an intervening
sequence to ensure high level expression. Expression can be
monitored by SDS-PAGE and staining, if expression levels are
sufficiently high. Additionally, if the protein is produced with a
tag, detection by anti-tag antibody can be carried out and if
produced with no tag, detection by anti-Hsp antibody that does not
recognize homologous proteins of the host may be employed. Further,
any method known in the art for protein identification may be
utilized to this end (e.g., a high resolution electrophoretic
method or 2D electrophoresis).
[0125] C. Preparation of Antibodies Against the Hsp Polypeptides of
the Present Invention
[0126] In another aspect, the proteins of the present invention are
utilized to prepare specifically binding antibodies (i.e., binding
partners). Accordingly, the present invention also provides such
antibodies. Within the context of the present invention, the term
"antibodies" includes polyclonal antibodies, monoclonal antibodies,
anti-idiotypic antibodies, fragments thereof such as F(ab').sub.2
and Fab fragments, and recombinantly or synthetically produced
binding partners. Such binding partners incorporate the variable
regions that permit an antibody to specifically bind, which means
an antibody able to selectively bind to a peptide produced from one
of the Neisserial, Aspergillal or Candidal Hsp genes of the
invention with a Kd of about 10.sup.-3 M or less. The affinity of
an antibody or binding partner can be readily determined by one of
ordinary skill in the art (see Scatchard, Ann. N.Y Acad. Sci.
51:660-672, 1949).
[0127] Polyclonal antibodies can be readily generated by one of
ordinary skill in the art from a variety of warm-blooded animals
such as horses, cows, goats, sheep, dogs, chickens, turkeys,
rabbits, mice, or rats. Briefly, the desired protein or peptide is
utilized to immunize the animal through intraperitoneal,
intramuscular, intraocular, or subcutaneous injections. The
immunogenicity of the protein or peptide of interest may be
increased through the use of an adjuvant such as Freund's complete
or incomplete adjuvant. Following several booster immunizations,
small samples of serum are collected and tested for reactivity to
the desired protein or peptide.
[0128] Typically, suitable polyclonal antisera give a signal that
is at least three times greater than background. Once the titer of
the animal has reached a plateau in terms of its reactivity to the
protein, larger quantities of polyclonal antisera may be readily
obtained either by weekly bleedings, or by exsanguinating the
animal.
[0129] Monoclonal antibodies can also be readily generated using
well-known techniques (see U.S. Pat. Nos. RE 32,011, 4,902,614,
4,543,439, and 4,411,993; see also Monoclonal Antibodies,
Hybridomas: A New Dimension in Biological Analyses, Plenum Press,
Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A
Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor
Laboratory Press, 1988). Briefly, in one embodiment, a subject
animal such as a rat or mouse is injected with a desired protein or
peptide. If desired, various techniques may be utilized in order to
increase the resultant immune response generated by the protein, in
order to develop greater antibody reactivity. For example, the
desired protein or peptide may be coupled to another protein such
as ovalbumin or keyhole limpet hemocyanin (KLH), or through the use
of adjuvants such as Freund's complete or incomplete adjuvant. The
initial elicitation of an immune response, may preferably be
through intraperitoneal, intramuscular, intraocular, or
subcutaneous routes.
[0130] Between one and three weeks after the initial immunization,
the animal may be reimmunized. The animal may then be test bled and
the serum tested for binding to the desired antigen using assays as
described above. Additional immunizations may also be accomplished
until the animal has reached a plateau in its reactivity to the
desired protein or peptide. The animal may then be given a final
boost of the desired protein or peptide, and three to four days
later sacrificed. At this time, the spleen and lymph nodes may be
harvested and disrupted into a single cell suspension by passing
the organs through a mesh screen or by rupturing the spleen or
lymph node membranes which encapsulate the cells. Within one
embodiment the red cells are subsequently lysed by the addition of
a hypotonic solution, followed by immediate return to
isotonicity.
[0131] Within another embodiment, suitable cells for preparing
monoclonal antibodies are obtained through the use of in vitro
immunization techniques. Briefly, an animal is sacrificed, and the
spleen and lymph node cells are removed as described above. A
single cell suspension is prepared, and the cells are placed into a
culture containing a form of the protein or peptide of interest
that is suitable for generating an immune response as described
above. Subsequently, the lymphocytes are harvested and fused as
described below.
[0132] Cells that are obtained through the use of in vitro
immunization or from an immunized animal as described above may be
immortalized by transfection with a virus such as the Epstein-Barr
Virus (EBV). (See Glasky and Reading, Hybridoma 8(4):377-389,
1989.) Alternatively, within a preferred embodiment, the harvested
spleen and/or lymph node cell suspensions are fused with a suitable
myeloma cell in order to create a "hybridoma" which secretes
monoclonal antibodies. Suitable myeloma lines are preferably
defective in the construction or expression of antibodies, and are
additionally syngeneic with the cells from the immunized animal.
Many such myeloma cell lines are well known in the art and may be
obtained from sources such as the American Type Culture Collection
(ATCC), Rockville, Md. (see Catalogue of Cell Lines &
Hybridomas, 6.sup.th ed., ATCC, 1988). Representative myeloma lines
include: for humans, UC 729-6 (ATCC No. CRL 8061), MC/CAR-Z2 (ATCC
No. CRL 8147), and SKO-007 (ATCC No. CRL 8033); for mice,
SP2/0-Ag14 (ATCC No. CRL 1581), and P3X63Ag8 (ATCC No. TIB 9); and
for rats, Y3-Ag1.2.3 (ATCC No. CRL 1631), and YB2/0 (ATCC No. CRL
1662). Particularly preferred fusion lines include NS-1 (ATCC No.
TIB 18) and P3X63--Ag 8.653 (ATCC No. CRL 1580), which may be
utilized for fusions with either mouse, rat, or human cell lines.
Fusion between the myeloma cell line and the cells from the
immunized animal can be accomplished by a variety of methods,
including the use of polyethylene glycol (PEG) (see Antibodies: A
Laboratory Manual, Harlow and Lane, supra) or electrofusion. (See
Zimmerman and Vienken, J. Membrane Biol. 67:165-182, 1982.)
[0133] Following the fusion, the cells are placed into culture
plates containing a suitable medium, such as RPMI 1640 or DMEM
(Dulbecco's Modified Eagles Medium, JRH Biosciences, Lenexa,
Kans.). The medium may also contain additional ingredients, such as
Fetal Bovine Serum (FBS, e.g., from Hyclone, Logan, Utah, or JRH
Biosciences), thymocytes that were harvested from a baby animal of
the same species as was used for immunization, or agar to solidify
the medium. Additionally, the medium should contain a reagent which
selectively allows for the growth of fused spleen and myeloma
cells. Particularly preferred is the use of HAT medium
(hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co., St.
Louis, Mo.). After about seven days, the resulting fused cells or
hybridomas may be screened in order to determine the presence of
antibodies which recognize the desired antigen. Following several
clonal dilutions and reassays, hybridoma producing antibodies that
bind to the protein of interest can be isolated.
[0134] Other techniques may also be utilized to construct
monoclonal antibodies. (See Huse et al., "Generation of a Large
Combinational Library of the Immunoglobulin Repertoire in Phage
Lambda," Science 246:1275-1281, 1989; see also Sastry et al.,
"Cloning of the Immunological Repertoire in Escherichia coli for
Generation of Monoclonal Catalytic Antibodies: Construction of a
Heavy Chain Variable Region-Specific cDNA Library," Proc. Natl.
Acad. Sci. USA 86:5728-5732, 1989; see also Alting-Mees et al.,
"Monoclonal Antibody Expression Libraries: A Rapid Alternative to
Hybridomas," Strategies in Molecular Biology 3:1-9, 1990; these
references describe a commercial system available from Stratagene,
La Jolla, Calif., which enables the production of antibodies
through recombinant techniques.) Briefly, mRNA is isolated from a B
cell population and utilized to create heavy and light chain
immunoglobulin cDNA expression libraries in the
.lambda.IMMUNOZAP(H) and .lambda.IMMUNOZAP(L) vectors. These
vectors may be screened individually or co-expressed to form Fab
fragments or antibodies (see Huse et al. (supra); see also Sastry
et al. (supra)).
[0135] Similarly, binding partners can also be constructed
utilizing recombinant DNA techniques to incorporate the variable
regions of a gene that encodes a specifically binding antibody. The
construction of these binding partners can be readily accomplished
by one of ordinary skill in the art given the disclosure provided
herein. (See Larrick et al., "Polymerase Chain Reaction Using Mixed
Primers: Cloning of Human Monoclonal Antibody Variable Region Genes
From Single Hybridoma Cells," Biotechnology 7:934-938, 1989,
Riechmann et al., "Reshaping Human Antibodies for Therapy," Nature
332:323-327, 1988; Roberts et al., "Generation of an Antibody with
Enhanced Affinity and Specificity for its Antigen by Protein
Engineering," Nature 328:731-734, 1987; Verhoeyen et al.,
"Reshaping Human Antibodies: Grafting an Antilysozyme Activity,"
Science 239:1534-1536, 1988; Chaudhary et al., "A Recombinant
Immunotoxin Consisting of Two Antibody Variable Domains Fused to
Pseudomonas Exotoxin." Nature 339:394-397, 1989; see also U.S. Pat.
No. 5,132,405 entitled "Biosynthetic Antibody Binding Sites.")
Briefly, in one embodiment, DNA segments encoding the desired
protein or peptide of interest-specific antigen binding domains are
amplified from hybridomas that produce a specifically binding
monoclonal antibody, and are inserted directly into the genome of a
cell that produces human antibodies. (See Verhoeyen et al. (supra);
see also Reichmann et al. (supra)). This technique allows the
antigen-binding site of a specifically binding mouse or rat
monoclonal antibody to be transferred into a human antibody. Such
antibodies are preferable for therapeutic use in humans because
they are not as antigenic as rat or mouse antibodies.
[0136] In an alternative embodiment, genes that encode the variable
region from a hybridoma producing a monoclonal antibody of interest
are amplified using oligonucleotide primers for the variable
region. These primers may be synthesized by one of ordinary skill
in the art, or may be purchased from commercially available
sources. For instance, primers for mouse and human variable regions
including, among others, primers for V.sub.Ha, V.sub.Hb, V.sub.Hc,
V.sub.Hd, C.sub.H1, V.sub.L and C.sub.L regions, are available from
Stratagene (La Jolla, Calif.). These primers may be utilized to
amplify heavy or light chain variable regions, which may then be
inserted into vectors such as IMMUNOZAP.TM.(H) or IMMUNOZAP.TM.(L)
(Stratagene), respectively. These vectors may then be introduced
into E. coli for expression. Utilizing these techniques, large
amounts of a single-chain polypeptide containing a fusion of the
V.sub.H and V.sub.L domains may be produced (see Bird et al.,
Science 242:423-426, 1988).
[0137] Monoclonal antibodies and other binding partners can be
produced in a number of host systems, including tissue cultures,
bacteria, eukaryotic cells, plants and other host systems in the
art.
[0138] Once suitable antibodies or binding-partners have been
obtained, they may be isolated or purified by many techniques well
known to those of ordinary skill in the art (see Antibodies: A
Laboratory Manual, Harlow and Lane (supra)). Suitable techniques
include peptide or protein affinity columns, HPLC or RP-HPLC,
purification on protein A or protein G columns, or any combination
of these techniques. Within the context of the present invention,
the term "isolated" as used to define antibodies or binding
partners means "substantially free of other blood components."
[0139] The binding partners of the present invention have many
uses. For example, antibodies can be utilized in flow cytometry to
identify cells bearing such a protein. Briefly, in order to detect
the protein or peptide of interest on cells, the cells are
incubated with a labeled monoclonal antibody which specifically
binds to the protein of interest, followed by detection of the
presence of bound antibody. Labels suitable for use within the
present invention are well known in the art including, among
others, flourescein isothiocyanate (FITC), phycoerythrin (PE),
horse radish peroxidase (HRP), and colloidal gold. Particularly
preferred for use in flow cytometry is FITC, which may be
conjugated to purified antibody according to the method of Keltkamp
in "Conjugation of Fluorescein Isothiocyanate to Antibodies. I.
Experiments on the Conditions of Conjugation," Immunology
18:865-873, 1970. (See also Keltkamp, "Conjugation of Fluorescein
Isothiocyanate to Antibodies. II. A Reproducible Method,"
Immunology 18:875-881, 1970; Goding, "Conjugation of Antibodies
with Fluorochromes: Modification to the Standard Methods," J.
Immunol. Methods 13:215-226, 1970.) The antibodies can also be used
to target drugs to Neisseria meningitidis, Aspergillus fumigatus or
Candida glabrata as well as a diagnostic for determining infection
by these organisms.
[0140] D. Assays that Utilize the Hsp Polypeptides, or Antibodies
Thereto, of the Present Invention
[0141] A variety of assays can be utilized in order to detect the
Hsp polypeptides from Neisseria meningitidis, Aspergillus fumigatus
or Candida glabrata of the present invention, or antibodies that
specifically bind to such Hsp polypeptides. Exemplary assays are
described in detail in Antibodies: A Laboratory Manual, Harlow and
Lane (eds.), Cold Spring Harbor Laboratory Press, 1988.
Representative examples of such assays include: countercurrent
immuno-electrophoresis (CIEP), radioimmunoassays,
radioimmunoprecipitations, enzyme-linked immuno-sorbent assays
(ELISA), dot blot assays, inhibition or competition assays, and
sandwich-assays, immunostick (dipstick) assays, simultaneous
immunoassays, immunochromatographic assays, immunofiltration
assays, latex bead agglutination assays, immunofluorescent assays,
biosensor assays. and low-light detection assays (see U.S. Pat.
Nos. 4,376,110 and 4,486,530; see also Antibodies: A Laboratory
Manual (supra).
[0142] A fluorescent antibody test (FA-test) uses a fluorescently
labeled antibody able to bind to one of the proteins of the
invention. For detection, visual determinations are made by a
technician using fluorescence microscopy, yielding a qualitative
result. In one embodiment, this assay is used for the examination
of tissue samples or histological sections.
[0143] In latex bead agglutination assays, antibodies to one or
more of the proteins of the present invention are conjugated to
latex beads. The antibodies conjugated to the latex beads are then
contacted with a sample under conditions permitting the antibodies
to bind to desired proteins in the sample, if any. The results are
then read visually, yielding a qualitative result. In one
embodiment, this format can be used in the field for on-site
testing.
[0144] Enzyme immunoassays (EIA) include a number of different
assays able to utilize the antibodies provided by the present
invention. For example, a heterogeneous indirect EIA uses a solid
phase coupled with an antibody of the invention and an affinity
purified, anti-IgG immunoglobulin preparation. Preferably, the
solid phase is a polystyrene microtiter plate. The antibodies and
immunoglobulin preparation are then contacted with the sample under
conditions permitting antibody binding, which conditions are well
known in the art. The results of such an assay can be read
visually, but are preferably read using a spectrophotometer, such
as an ELISA plate reader, to yield a quantitative result. An
alternative solid phase EIA format includes plastic-coated ferrous
metal beads able to be moved during the procedures of the assay by
means of a magnet. Yet another alternative is a low-light detection
immunoassay format. In this highly sensitive format, the light
emission produced by appropriately labeled bound antibodies are
quantitated automatically. Preferably, the reaction is performed
using microtiter plates.
[0145] In an alternative embodiment, a radioactive tracer is
substituted for the enzyme mediated detection in an EIA to produce
a radioimmunoassay (RIA).
[0146] In a capture-antibody sandwich enzyme assay, the desired
protein is bound between an antibody attached to a solid phase,
preferably a polystyrene microtiter plate, and a labeled antibody.
Preferably, the results are measured using a spectrophotometer,
such as an ELISA plate reader.
[0147] In a sequential assay format, reagents are allowed to
incubate with the capture antibody in a step wise fashion. The test
sample is first incubated with the capture antibody. Following a
wash step, an incubation with the labeled antibody occurs. In a
simultaneous assay, the two incubation periods described in the
sequential assay are combined. This eliminates one incubation
period plus a wash step.
[0148] A dipstick/immunostick format is essentially an immunoassay
except that the solid phase, instead of being a polystyrene
microtiter plate, is a polystyrene paddle or dipstick. Reagents are
the same and the format can either be simultaneous or
sequential.
[0149] In a chromatographic strip test format, a capture antibody
and a labeled antibody are dried onto a chromatographic strip,
which is typically nitrocellulose or nylon of high porosity bonded
to cellulose acetate. The capture antibody is usually spray dried
as a line at one end of the strip. At this end there is an
absorbent material that is in contact with the strip. At the other
end of the strip the labeled antibody is deposited in a manner that
prevents it from being absorbed into the membrane. Usually, the
label attached to the antibody is a latex bead or colloidal gold.
The assay may be initiated by applying the sample immediately in
front of the labeled antibody.
[0150] Immunofiltration/immunoconcentration formats combine a large
solid phase surface with directional flow of sample/reagents, which
concentrates and accelerates the binding of antigen to antibody. In
a preferred format, the test sample is preincubated with a labeled
antibody then applied to a solid phase such as fiber filters or
nitrocellulose membranes or the like. The solid phase can also be
precoated with latex or glass beads coated with capture antibody.
Detection of analyte is the same as standard immunoassay. The flow
of sample/reagents can be modulated by either vacuum or the wicking
action of an underlying absorbent material.
[0151] A threshold biosensor assay is a sensitive, instrumented
assay amenable to screening large numbers of samples at low cost.
In one embodiment, such an assay comprises the use of light
addressable potentiometric sensors wherein the reaction involves
the detection of a pH change due to binding of the desired protein
by capture antibodies, bridging antibodies and urease-conjugated
antibodies. Upon binding, a pH change is effected that is
measurable by translation into electrical potential (.mu.volts).
The assay typically occurs in a very small reaction volume, and is
very sensitive. Moreover, the reported detection limit of the assay
is 1,000 molecules of urease per minute.
[0152] The present invention also provides for probes and primers
for detecting Neisseria meningitidis, Aspergillus fumigatus and
Candida glabrata.
[0153] In one embodiment of this aspect of the invention, probes
are provided that are capable of specifically hybridizing to
Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata
Hsp genes DNA or RNA. For purposes of the present invention, probes
are "capable of hybridizing" to Neisseria meningitidis, Aspergillus
fumigatus or Candida glabrata Hsp gene DNA or RNA if they hybridize
under conditions of high stringency (see Sambrook et al. (supra)).
Preferably, the probe may be utilized to hybridize to suitable
nucleotide sequences under highly stringent conditions, such as
6.times.SSC, 1.times.Denhardt's solution (Sambrook et al. (supra)),
0.1% SDS at 65.degree. C. and at least one wash to remove excess
probe in the presence of 0.2.times.SSC, 1.times.Denhardt's
solution, 0.1% SDS at 65.degree. C. Except as otherwise provided
herein, probe sequences are designed to allow hybridization to
Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata
DNA or RNA sequences, but not to DNA or RNA sequences from other
organisms, particularly other bacterial and fungal sequences. The
probes are used, for example, to hybridize to nucleic acids that
have been isolated from a test sample. The hybridized probe is then
detected, thereby indicating the presence of the desired cellular
nucleic acid. Preferably, the cellular nucleic acid is subjected to
an amplification procedure, such as PCR, prior to
hybridization.
[0154] Probes of the present invention may be composed of either
deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), and may be
as few as about 12 nucleotides in length or more typically about 18
to 24 nucleotides or longer comprising a sequence derived from a
fragment of the sequences of the Neisseria meningitidis,
Aspergillus fumigatus or Candida glabrata Hsp genes provided by
this invention. As used herein, a sequence is "derived from a
fragment" when it contains a nucleotide sequences identical to a
contiguous nucleotide sequence present in the fragment, or contains
a nucleotide sequence that results from reading errors that occur
during a PCR amplification of the fragment, or contains a
degenerate nucleotide sequence that encodes an amino acid sequence
that is identical to or has conservative substations of an amino
acid sequence encoded by the fragment. Selection of probe size is
somewhat dependent upon the use of the probe, and is within the
skill of the art.
[0155] Suitable probes can be constructed and labeled using
techniques that are well known in the art. Shorter probes of, for
example, 12 bases can be generated synthetically. Longer probes of
about 75 bases to less than 1.5 kb are preferably generated by, for
example, PCR amplification in the presence of labeled precursors
such as [.alpha.-.sup.32P]dCTP, digoxigenin-dUTP, or biotin-dATP.
Probes of more than 1.5 kb are generally most easily amplified by
transfecting a cell with a plasmid containing the relevant probe,
growing the transfected cell into large quantities, and purifying
the relevant sequence from the transfected cells. (See Sambrook et
al. (supra)).
[0156] Probes can be labeled by a variety of markers, including for
example, radioactive markers, fluorescent markers, enzymatic
markers, and chromogenic markers. The use of .sup.32P is
particularly preferred for marking or labeling a particular
probe.
[0157] It is a feature of this aspect of the invention that the
probes can be utilized to detect the presence of Neisseria
meningitidis, Aspergillus fumigatus or Candida glabrata Hsp mRNA or
DNA within a sample. However, if the organisms are present in only
a limited number, then it may be beneficial to amplify the relevant
sequence such that it may be more readily detected or obtained.
[0158] A variety of methods may be utilized in order to amplify a
selected sequence, including, for example, RNA amplification (see
Lizardi et al., Bio/Technology 6:1197-1202, 1988; Kramer et al.,
Nature 339:401-402, 1989; Lomeli et al., Clinical Chem.
35(9):1826-1831, 1989; U.S. Pat. No. 4,786,600), and DNA
amplification utilizing LCR or Polymerase Chain Reaction ("PCR")
(see U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; see also
U.S. Pat. Nos. 4,876,187 and 5,011,769, which describe an
alternative detection/amplification system comprising the use of
scissile linkages), or other nucleic acid amplification procedures
that are well within the level of ordinary skill in the art. With
respect to PCR, for example, the method may be modified as known in
the art. PCR may also be used in combination with reverse dot blot
hybridization (Iida et al., FEMS Microbiol. Lett. 114:167-172,
1993). PCR products may be quantitatively analyzed by incorporation
of dUTP (Duplas et al., Anal. Biochem. 212:229-236, 1993), and
samples may be filter sampled for PCR-gene probe detection (Bej et
al., Appl. Environ. Microbiol. 57:3529-3534, 1991).
[0159] Within a preferred embodiment, PCR amplification is utilized
to detect Neisseria meningitidis, Aspergillus fumigatus or Candida
glabrata Hsp DNA. Briefly a DNA sample is denatured at 95.degree.
C. in order to generate single-stranded DNA. Specific primers are
then annealed to the single-stranded DNA at 37.degree. C. to
70.degree. C., depending on the proportion of AT/GC in the primers.
The primers are extended at 72.degree. C. with Taq DNA polymerase
in order to generate the opposite strand to the template. These
steps constitute one cycle, which may be repeated in order to
amplify the selected sequence.
[0160] Within an alternative preferred embodiment, LCR
amplification is utilized for amplification. LCR primers are
synthesized such that the 5' base of the upstream primer is capable
of hybridizing to a unique sequence in a desired gene to
specifically detect a strain of Neisseria meningitidis, Aspergillus
fumigatus or Candida glabrata harboring the desired gene.
[0161] Within another preferred embodiment, the probes are used in
an automated, non-isotopic strategy wherein target nucleic acid
sequences are amplified by PCR, and then desired products are
determined by a colorimetric oligonucleotide ligation assay (OLA)
(Nickerson et al., Proc. Natl. Acad Sci. USA 81:8923-8927,
1990).
[0162] Primers for the amplification of a selected sequence should
be selected from sequences that are highly specific and form stable
duplexes with the target sequence. The primers should also be
non-complementary, especially at the 3' end, should not form dimers
with themselves or other primers, and should not form secondary
structures or duplexes with other regions of DNA. In general,
primers of about 18 to 20 nucleotides are preferred, and can be
easily synthesized using techniques well known in the art. PCR
products, and other nucleic acid amplification products, may be
quantitated using techniques known in the art (Dupla et al., Anal.
Biochem. 212:229-236, 1993; Higuchi et al., Bio/Technology
11:1026-1030).
[0163] Further a biochip array specific for Neisseria meningitidis,
Aspergillus fumigatus or Candida glabrata, comprised of a substrate
to which either oligonucleotides, polypeptides or antibodies may be
bound can be manufactured using the invention disclosed herein in
combination with current biochip technologies. U.S. Pat. No.
5,445,934. By using such a substrate with oligonucleotides derived
from the Hsp sequences of this invention or antibodies specific for
the Hsp gene products of this invention, a high throughput
screening tool can be created to identify the specific Neisseria,
Aspergillus or Candida species in many samples.
[0164] E. Pharmaceutical Compositions and Methods
[0165] By administering a Neisserial, Aspergillal or Candidal Hsp
to an animal, the respective Hsp can induce an immune response in
the animal to Neisseria, Aspergillus or Candida species,
respectively, preferably providing resistance to such bacterial or
fungal infection. Accordingly, the isolation of Neisserial,
Aspergillal and Candidal Hsp genes and polypeptides of the present
invention provides a platform for the generation of compositions
containing isolated polypeptides, fragments or variants of Hsps
that are useful in diagnosis and treatment of Neisserial,
Aspergillal or Candidal associated disorders. As used herein,
"treatment" means to administer an agent that prevents or reduces
the severity of a disorder caused by an infection by a Neisseria,
Aspergillus or Candida species.
[0166] Therefore, another aspect of the present invention provides
compositions and methods comprising one or more of the
above-described Hsp polypeptides or antibodies to Hsps in
combination with one or more pharmaceutically or physiologically
acceptable carriers, adjuvants, binders or diluents. Such
compositions can be used to elicit or enhance an immune response,
while antibodies can be used to block progression of disease in a
recipient animal, which is preferably a human being, and preferably
elicits or enhances a protective or partially protective immunity
against Neisseria meningitidis, Aspergillus fumigatus or Candida
glabrata, or against an organism that is targeted by an antigen
fused to an Hsp of the present invention.
[0167] Preferably, such carriers, adjuvants, binders or diluents
are nontoxic to recipients at the dosages and concentrations
employed. Ordinarily, the preparation of such compositions entails
combining the isolated Hsp polypeptide of this invention with
buffers, antioxidants such as ascorbic acid, low molecular weight
(less than about 10 residues) polypeptides, proteins, amino acids,
carbohydrates including glucose, sucrose or dextrins, chelating
agents such as EDTA, glutathione and other stabilizers and
excipients. Neutral buffered saline or saline mixed with
nonspecific serum albumin are exemplary appropriate diluents.
Examples of adjuvants include alum or aluminum hydroxide for
humans.
[0168] It will be evident in light of the present specification to
those in the art that the amount and frequency of administration
can be optimized in clinical trials, and will depend upon such
factors as the disease or disorder to be treated, the degree of
immune inducement, enhancement, or protection required, and many
other factors.
[0169] In one embodiment, the composition is administered orally,
and the purified Hsp of the invention is taken up by cells such as
cells located in the lumen of the gut. Alternatively, the Hsp
composition can be parenterally administrated via the subcutaneous
route, or via other parenteral routes. Other routes include
buccal/sublingual, rectal, nasal, topical (such as transdermal and
ophthalmic), vaginal, pulmonary, intraarterial, intramuscular,
intraperitoneal, intraocular, intranasal or intravenous, or
indirectly. The Hsp compositions of the present invention can be
prepared and administered as a liquid solution, or prepared as a
solid form (e.g., lyophilized) which can be administered in solid
form or resuspended in a solution in conjunction with
administration.
[0170] Depending upon the application, quantities of injected Hsp
in the composition will vary generally from about 0.1 .mu.g to 1000
mg, typically from about 1 .mu.g to 100 mg, preferably from about
10 .mu.g to 10 mg, and preferably from about 100 .mu.g to 1 mg, in
combination with the physiologically acceptable carrier, binder or
diluent. Booster immunizations can be given from 2-6 weeks
later.
[0171] The pharmaceutical compositions of the present invention may
be placed within containers, along with packaging material,
preferably consumer-acceptable, which provides instructions
regarding the use of such pharmaceutical compositions, to provide
kits suitable for use within the present invention. Generally, such
instructions will include a tangible expression describing the
reagent concentration, as well as within certain embodiments,
relative amounts of excipient ingredients or diluents (e.g., water,
saline or PBS) which may be necessary to reconstitute the
pharmaceutical composition.
[0172] The Hsp gene products of this invention may also be used as
immunological carriers in conjugate vaccines. Hsps are beneficial
carriers of antigens because, unlike other carriers, they do not
have an immunosuppressive effect. See Barrios et al., Eur. J.
Immunol. 22:1365-1372, 1992; Suzue and Young, in Stress-Inducible
Cellular Responses 77:451-465, 1996 (edited by U. Feige et al.).
Such carriers may be used to elicit an increased immune response to
the conjugated molecule. Alternatively, small Hsp peptides or
polypeptides containing antigenic epitopes derived from the larger
Hsp polypeptides provided herein, can be conjugated or fused to
other carrier proteins to elicit an immunogenic response to the
small Hsp antigenic epitope. The Hsp gene products of this
invention may therefore be used (in conjugates or fusion proteins)
as carriers to elicit an immunogenic response against other target
antigens, or as antigens to elicit an immunogenic response against
epitopes present on the Hsps.
[0173] As used herein, a "fusion protein" is a protein comprised of
a Hsp polypeptide, or portion thereof. which is has a peptide bond
linkage with an amino acid sequence of an additional polypeptide
chain such that a single polypeptide chain is formed which contains
an amino acid sequence derived from the Hsp joined with an amino
acid sequence derived from the additional polypeptide chain. In one
example of typical fusion proteins, a Hsp polypeptide or portion
thereof is fused to an additional carrier polypeptide that enhances
an immunogenic response in an animal. Example of such polypeptides
include but are not limited to keyhole limpet hemocyanin, (KLH)
bovine gamma globulin (BGG), serum albumin from various animals
(SA) and polypeptides that provide antigenic determinants in
addition to that provided by a single Hsp domain. Additional
antigenic determinants may include for example, polypeptide domains
from more than one Hsp and/or multiple duplications of an antigenic
polypeptide domain from a single Hsp. In these cases the target of
the immune response of interest is typically the Hsp portion of the
fusion protein. Alternatively, a Hsp polypeptide of the present
invention can serve as the carrier portion of a fusion polypeptide
when the additional polypeptide to which it is fused is the target
antigen for eliciting an immunogenic response.
[0174] One method for producing a fusion protein is by in frame
ligation of a nucleic acid sequence encoding a Hsp with a nucleic
acid sequence encoding an additional polypeptide to form a hybrid
sequence. The hybrid sequence is inserted into an expression vector
to form a construct having the hybrid fragment under the control of
a promoter operably linked thereto. The construct is introduced
into a suitable host cell capable of expressing the hybrid fragment
from the vector sequence. Upon expression, the fusion protein is
produced and may be isolated from the host cell. When the host cell
is a bacterium, the fusion protein may aggregate into inclusion
bodies and be readily isolated using methods well known in the art.
Alternatively, the fusion protein may include signaling sequences
selected to direct the fusion protein to export from the cell into
the extracellular medium in which the host cell is cultured.
[0175] A further aspect of the present invention is protection from
Neisserial, Aspergillal or Candidal associated diseases by either
immunization with the Hsp gene products of the present invention
(e.g. by intramuscular injection of an expression vector containing
an Hsp gene) or by using gene transfer techniques to deliver a
vector containing Hsp genes or fragments thereof to be expressed
within the cells of the animal.
[0176] The compositions and methodologies described herein are
suitable for a variety of uses. To this end, the following examples
are presented for purposes of illustration, not limitation.
EXAMPLES
Example 1
Cloning of an Internal Fragment of the Neisseria meningitidis Hsp70
Gene
[0177] Comparison of previously characterized bacterial Hsp70 (or
DnaK) proteins was used to identify conserved regions and design
degenerate primers suitable for PCR-amplification of an internal
region of the unknown Neisseria meningitidis Hsp70 gene.
[0178] Forward degenerate primer W247 corresponded to a sequence
encoding amino acids 145-150 of the consensus Hsp70 sequence
(PAYFND).
[0179] W247: 5'-CCNGCNTAYTTYAAYGAY-3'
[0180] Reverse degenerate primer W248 was complementary to a
sequence encoding amino acids 476-482 of the consensus Hsp70
sequence (PQIEVTF).
[0181] W248: 5'-RAANGTNACYTCDATYTGNGG-3'
[0182] Note that in all sequences provided herein A corresponds to
adenosine, C to cytidine, G to guanosine, T to thymidine, I to
inosine, R to A or G, Y to C or T, N to A, C, T or G, K to G or T,
M to A or C, S to G or C, W to T or A, B to C, G or T, D to A, G or
T, H to A, C or T, and V to A, C or G. Unless specified, all
molecular DNA manipulations (plasmid isolation, restriction enzyme
digestion, ligation, etc.) were carried on under standard
conditions described in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, 1989.
[0183] PCR reactions were carried out according to Perkin-Elmer's
recommendations. All reagents used for PCR reactions were supplied
by Perkin-Elmer unless indicated otherwise. Reaction mixtures
(total volume of 100 ul) contained 0.5 to 1 ug of genomic DNA, 100
pmoles of each of the degenerate primers (W247 and W248,
synthesized by Life Technologies), 500 uM each of dNTPs (New
England BioLabs), 1.times.PCR buffer, 4 mM MgSO4, and 1.25 units of
Taq polymerase. Reactions were incubated at 95.degree. C. for 30
seconds, at 51.degree. C. for 3 minutes and then at 72.degree. C.
for 1 minute. After repeating the above cycle for a total of 40
times, reactions were incubated at 72.degree. C. for an additional
4 minutes. Genomic DNA from Neisseria meningitidis (ATCC-13090) was
obtained from Dr. Lee Weber (University of Nevada, Reno, Nev.,
USA).
[0184] A PCR fragment of about one kbp in length was isolated from
a low-melting point agarose gel by phenol extraction and ligated
into pCR2.1 TA cloning vector (Invitrogen) using standard
conditions. The ligation reaction was used to transform E. coli
DH5a cells, and transformants were selected on LB agar plates with
kanamycin D. Recombinant plasmids were identified after digestion
of plasmid DNA with EcoRI restriction enzyme.
[0185] Plasmids containing the above fragment of Neisseria
meningitidis Hsp70 gene were subjected to DNA sequencing using the
dye-terminator method on a Prizm 310 automatic sequencer (ABI).
Sequence data were assembled and analyzed using DNA Star (DNASTAR
Inc.) as well as DNA Strider (CEA, France) software. E. coli dnaK
gene and protein sequences available from GenBank were used for
comparison purposes during assembly.
[0186] Three clones originating from a mixture of three separate
PCR reactions were sequenced using M13 forward and reverse
universal primers:
1 M13F: 5'-GTAAAACGACGGCCAG-3' M13R: 5'-CAGGAAACAGCTATGAC-3'
[0187] Sequences obtained were used to design an additional pair of
primers for sequencing:
2 N1: 5'-CTGCCGTACATCACCATGG-3' N2: 5'-GGCTTCTTGTACTTTCGGC-3'
[0188] FIG. 1 shows the strategy for sequencing the internal
Neisseria meningitidis Hsp70 gene fragment.
[0189] FIG. 2 lists the DNA sequence of this region (assembled from
information obtained from sequencing three Hsp70 gene
fragment-containing plasmids).
Example 2
Cloning the Ends of the Neisseria meningitidis Hsp70 Gene by
Inverse PCR
[0190] The so called inverse PCR approach was used to clone missing
ends of the Neisseria meningitidis Hsp70 gene. From the restriction
map of the assembled partial DNA sequence BamHI, EcoRI, HincII and
Hind III were chosen as enzymes that do not cut.
[0191] Approximately 2 ug of Neisseria meningitidis genomic DNA
were digested with each of the above enzymes, phenol-extracted,
precipitated with ethanol and dissolved in 1.times.ligation buffer
at approx. 80 ng/ml. Fragments were ligated and then used as
templates for PCR amplification (40 cycles of 1 minute at
95.degree. C., 2 minutes at 65.degree. C. and 2 minutes at
72.degree. C.) in reaction mixtures -(as described above)
containing primers nested near the ends of known sequence and
pointing outside:
3 N70-5: 5'-GGTCGGCTCGTTGATGATGCGTTTCAC-3' N70-3:
5'-GCTTCTGCCAACAAATCTTTGGGTCAG-3'
[0192] Only DNA digested with HindIII seemed to produce a specific
PCR-generated band after amplification. The 0.9 kbp-long fragment
was purified from a low-melting point agarose gel and cloned into
pCR2.1 vector. A recombinant containing the fragment was
identified, and the inserted fragment was sequenced using M13F and
M13R primers. It turned out that cloned fragment had been amplified
making use of only the N70-5 primer. Still, it represented a region
from the 5' end of the Neisseria meningitidis Hsp70 gene.
Unfortunately, only 44 nucleotides of new sequence could be
determined because a HindIII site happened to be present just
upstream from the 5'-end of the previously sequenced internal Hsp70
gene fragment. Nevertheless, the sequence of the fragment allowed
the resolution of ambiguities created by the use of the degenerate
W247 primer. See region B in FIG. 3 as well as the sequence in FIG.
4.
[0193] A Neisseria meningitidis (ATCC13090) genomic library in
bacteriophage lambda was prepared using routine procedures and was
screened using the above-described internal region of the Neisseria
meningitidis Hsp70 gene as the probe. A recombinant clone
containing the Neisseria meningitidis Hsp70 gene was used as
template for inverse PCR. Additional primers (pointing towards the
interior of the known Hsp70 sequence) were designed near the known
ends of the internal Hsp70 fragment in regions not interrupted by
RsaI restriction sites. Recombinant phage DNA was digested with
RsaI, and resulting fragments were circularized as above.
[0194] To amplify the 5'-end region, primers N70-5 (see above) and
N70-5B were used for PCR amplification of the ligation
reaction:
[0195] N-70-5B: 5'-GCCGCTTTGGCATTCGTTATGGAC-3'
[0196] To amplify the 3'-end region, primers N70-3 (see above) and
N70-3B were used for PCR of the ligation reaction:
[0197] N70-3B: 5'-GCGTTCGCGTTCGCCTTGCAGTAC-3'
[0198] PCR reactions were carried out as described before. PCR
products were isolated from low-melting point agarose gels and
inserted into vector pCR2.1 as also described before.
[0199] Sequencing of cloned PCR products revealed the complete
sequence of the 3' end of the Neisseria meningitidis Hsp70 gene
(and clarification of ambiguities resulting from the use of
degenerate primer W247 in the cloning of the internal Hsp70 region)
as well as 3'-untranslated sequence (region E in FIG. 3). The
nucleotide sequence of the 5' end of the Hsp70 gene was also
established (region C in FIG. 3), except for the first 28 bp (which
had been removed by RsaI digestion).
[0200] To determine the nucleotide sequence at the 5' end of the
Hsp70 gene, recombinant phage DNA was digested with restriction
enzyme Sau3A, fragments were circularized as before, and the
ligation reaction was subjected to PCR using a set of primers
located between 5' end of the known Hsp70 sequence and closest
internal Sau3A site:
4 N70-5C: 5'-TTCCGAAAACGGTCAAAC-3' N70-5D:
5'-ATGGCCAAACAAGAGTTG-3'
[0201] PCR reaction, isolation of PCR product from a low-melting
point agarose gel and ligation into vector pCR2.1 were carried out
as described before. The ligation reaction was then PCR-amplified
using M13F and N70-5C primers. The resulting PCR product was then
purified from an agarose gel using a gel extraction kit from Qiagen
and used directly for sequencing using the T7PROM primer. This
protocol produced the complete nucleotide of the 5'-end of the
Neisseria meningitidis Hsp70 gene as well as 5'-untranslated and
promoter sequences (region D in FIG. 3).
[0202] FIG. 4 lists the complete nucleotide sequence of the
Neisseria meningitidis Hsp70 gene as well as of flanking regions.
The derived amino acid sequence of the protein product of the gene
is also shown.
Example 3
Neisseria meningitidis Hsp70 Expression Vectors
[0203] To clone the Neisseria meningitidis Hsp70-coding region, DNA
from a recombinant bacteriophage containing the Hsp70 gene served
as the template in a PCR amplification reaction that included
primers N70-M and N70-Z, complementary to sequence at the 5'-end
(including an NdeI site) and the 3'-end of the Hsp70 gene,
respectively.
5 N70-M: 5'-TACATATGGCAAAAGTAATCGGTATC-3' N70-Z:
5'-TTTATTTTTTGTCGTCTTTTAC-3'
[0204] PCR product was purified from an agarose gel using a gel
extraction kit (Qiagen) and ligated into pCR2.1 vector. Two
positive clones were identified by EcoRI digestion of miniprep DNA
isolated from E. coli DH5a colonies resistant to kanamycin D and
further confirmed by restriction analysis using HindIII, NotI, NdeI
and ClaI. Inserted DNA was then sequenced using primers M13F, M13R,
N1, N2, N70-5, N70-5B, N70-5C, N70-3, N70-3B, as well as new primer
N10:
[0205] N10: 5'-GTCCAAATAAGCGATAACG-3'
[0206] FIG. 5 illustrates the sequencing strategy employed.
[0207] The sequence obtained (FIG. 6) differed from that presented
in FIG. 3 by an A instead of a G at positions 1528 (counted from
the NdeI recognition site) and 1647. Only the first of these
differences would also be reflected at the protein level. Because
sequence comparisons showed that residue 509 is typically serine,
the sequence presented in FIG. 6 was assumed to be the correct
sequence.
[0208] An NdeI--EcoRI (site located downstream from the stop codon)
fragment of the above pCR2.1-based plasmid including the complete
Hsp70 -coding sequence was inserted in between the NdeI and EcoRI
sites of pET24A+ and pET28A+ T7 expression vectors. Positive clones
were identified by digestion of DNA isolated from kanamycin
resistant transformed DH5a colonies with NdeI and EcoRI and
electrophoretic analysis. Single positive clones from each set was
sequenced using primers T7PROM, T7TERM, N1, N2, N70-5, N70-5B,
N70-5C, N70-3B and new primers
Sequence CWU 1
1
102 1 2465 DNA Nesseria meningitidis 1 caattcaaca tactgaacgc
caaagatatg gatgcagaac aagtctccct ttccaaagaa 60 tgcgacatca
tcgagtcttc acacgactgg gaaaaagagt acggcaactt gaacgaacag 120
gaaatgctcg ccggcatcgt ctatgaataa acctgcctgc catttgaaac attatgcttg
180 aatgcattgg agccaaatgt attaaatcaa atataaaacc aatatattca
taaagttata 240 tacttatagc catgctgtag cttgaaacag ttccaacata
cccgccgccc gccctattta 300 cagcccatcg ggacaaaatg tttctcaaaa
taagcaaaat caaataggat tatccacatg 360 gcaaaagtaa tcggtatcga
cttaggtaca accaactctt gtttggccat ttccgaaaac 420 ggtcaaacca
aagtgatcga aaacgcagaa ggcgcacgca ccacgccgtc cgttatcgct 480
tatttggacg gcggcgaaat cctcgtcggt gcgcctgcca aacgccaagc ggtaaccaac
540 gccaaaaaca ccatttacgc cgccaaacgt ttgatcggcc acaaatttga
agacaaagaa 600 gtccaacgcg acatcgaatc tatgcctttc gaaatcatca
aagccaacaa cggcgacgca 660 tgggtaaaag cacaaggcaa agagctgtct
cctcctcaaa tttccgcaga agtcctgcgt 720 aaaatgaaag aagccgccga
agcttacttg ggcgaaaaag taaccgaagc cgtgattacc 780 gtccctgcct
acttcaacga cagccaacgt caagccacca aagacgcagg ccgtatcgcc 840
ggtttggacg tgaaacgcat catcaacgag ccgaccgcag ccgctttggc attcggtatg
900 gacaaaggcg acaacaaaga ccgcaaagta gccgtatatg acttgggcgg
cggtactttc 960 gatatttcca tcatcgaaat cgccaacctc gacggcgaca
aacaattcga agtattggca 1020 accaacggcg ataccttctt gggcggtgaa
gacttcgacc aacgcctcat cgaccacatc 1080 atcgccgagt tcaaaaaaga
acaaggcatt gatttgaaac aagacgtgat ggctctacaa 1140 cgcctgaaag
aagctgccga aaaagccaaa atcgaattgt ccagcggcca gcaaaccgaa 1200
attaacctgc cgtacatcac catggacgca accggcccga aacacttggc gatgaaaatt
1260 acccgcgcca aattcgaaag cctggttgaa gacctgatta cccgctctat
cgaaccttgc 1320 aaaattgcat tgaaagatgc cggcttgagc accggcgaca
tcgacgacgt aatcttggtc 1380 ggcgggcagt cccgtatgcc gaaagtacaa
gaagccgtta aagccttctt cggcaaagaa 1440 ccgcgcaaag acgtgaaccc
tgacgaagcc gttgccgtag gcgcagcgat ccaaggcgaa 1500 gtattgagcg
gcggccgcag cgacgtattg ctactggacg taactcctct gtctttgggt 1560
atcgaaacca tgggcggcgt gatgaccaaa ctgattcaga agaacaccac catcccgacc
1620 aaagcgtcgc aagtgttctc taccgccgaa gacaaccaaa gcgcagtaac
catccacgta 1680 ctgcaaggcg aacgcgaacg cgcttctgcc aacaaatctt
tgggtcagtt caacttgggc 1740 gacatcgcac ctgcaccgcg cggtatgccg
caaatcgaag taaccttcga catcgacgcc 1800 aacggcatcc tgcacgtttc
cgccaaagac aaaggcaccg gtaaagcagc caacatcacc 1860 atccaaggtt
cttcaggttt gggcgaagaa gaaatcgaac gcatggtgaa agatgccgaa 1920
gccaatgccg aggaagataa aaaactgact gaattggtcg cttcccgcaa ccaagccgaa
1980 gccctgattc actctgtgaa gaaatctttg gccgactacg gcgacaaact
cgatgcagcc 2040 gagaaagaaa aaattgaagc cgcgctgaaa gaagccgaag
aagcagttaa aggcgacgac 2100 aaagccgcta tcgatgccaa aaccgaggcg
ctgggcgcag ccagccaaaa actgggggaa 2160 atggtttacg ctcaagcaca
agctgaagcc caagcaggcg aaagcgaaca agccaatgct 2220 tctgcaaaga
aagacgatga tgtcgtagat gccgactttg aagaagtaaa agacgacaaa 2280
aaataattaa taccgtctga aaaaagcgcg aaccgtttga ttcgcgcttt tttcaattga
2340 gataaaagac catagcataa cagaggttcc aagttcatct accgtaatat
tcccaaaccc 2400 aggttccaac tgttgtatcg gttctctgga attttttata
tagtggatta aatttaaacc 2460 agtac 2465 2 1929 DNA Nesseria
meningitidis 2 atggcaaaag taatcggtat cgacttaggt acaaccaact
cttgtttggc catttccgaa 60 aacggtcaaa ccaaagtgat cgaaaacgca
gaaggcgcac gcaccacgcc gtccgttatc 120 gcttatttgg acggcggcga
aatcctcgtc ggtgcgcctg ccaaacgcca agcggtaacc 180 aacgccaaaa
acaccattta cgccgccaaa cgtttgatcg gccacaaatt tgaagacaaa 240
gaagtccaac gcgacatcga atctatgcct ttcgaaatca tcaaagccaa caacggcgac
300 gcatgggtaa aagcacaagg caaagagctg tctcctcctc aaatttccgc
agaagtcctg 360 cgtaaaatga aagaagccgc cgaagcttac ttgggcgaaa
aagtaaccga agccgtgatt 420 accgtccctg cctacttcaa cgacagccaa
cgtcaagcca ccaaagacgc aggccgtatc 480 gccggtttgg acgtgaaacg
catcatcaac gagccgaccg cagccgcttt ggcattcggt 540 atggacaaag
gcgacaacaa agaccgcaaa gtagccgtat atgacttggg cggcggtact 600
ttcgatattt ccatcatcga aatcgccaac ctcgacggcg acaaacaatt cgaagtattg
660 gcaaccaacg gcgatacctt cttgggcggt gaagacttcg accaacgcct
catcgaccac 720 atcatcgccg agttcaaaaa agaacaaggc attgatttga
aacaagacgt gatggctcta 780 caacgcctga aagaagctgc cgaaaaagcc
aaaatcgaat tgtccagcgg ccagcaaacc 840 gaaattaacc tgccgtacat
caccatggac gcaaccggcc cgaaacactt ggcgatgaaa 900 attacccgcg
ccaaattcga aagcctggtt gaagacctga ttacccgctc tatcgaacct 960
tgcaaaattg cattgaaaga tgccggcttg agcaccggcg acatcgacga cgtaatcttg
1020 gtcggcgggc agtcccgtat gccgaaagta caagaagccg ttaaagcctt
cttcggcaaa 1080 gaaccgcgca aagacgtgaa ccctgacgaa gccgttgccg
taggcgcagc gatccaaggc 1140 gaagtattga gcggcggccg cagcgacgta
ttgctactgg acgtaactcc tctgtctttg 1200 ggtatcgaaa ccatgggcgg
cgtgatgacc aaactgattc agaagaacac caccatcccg 1260 accaaagcgt
cgcaagtgtt ctctaccgcc gaagacaacc aaagcgcagt aaccatccac 1320
gtactgcaag gcgaacgcga acgcgcttct gccaacaaat ctttgggtca gttcaacttg
1380 ggcgacatcg cacctgcacc gcgcggtatg ccgcaaatcg aagtaacctt
cgacatcgac 1440 gccaacggca tcctgcacgt ttccgccaaa gacaaaggca
ccggtaaagc agccaacatc 1500 accatccaag gttcttcagg tttgagcgaa
gaagaaatcg aacgcatggt gaaagatgcc 1560 gaagccaatg ccgaggaaga
taaaaaactg actgaattgg tcgcttcccg caaccaagcc 1620 gaagccctga
ttcactctgt gaaaaaatct ttggccgact acggcgacaa actcgatgca 1680
gccgagaaag aaaaaattga agccgcgctg aaagaagccg aagaagcagt taaaggcgac
1740 gacaaagccg ctatcgatgc caaaaccgag gcgctgggcg cagccagcca
aaaactgggg 1800 gaaatggttt acgctcaagc acaagctgaa gcccaagcag
gcgaaagcga acaagccaat 1860 gcttctgcaa agaaagacga tgatgtcgta
gatgccgact ttgaagaagt aaaagacgac 1920 aaaaaataa 1929 3 1929 DNA
Nesseria meningitidis 3 atggcaaaag taatcggtat cgacttaggt acaaccaact
cttgtttggc catttccgaa 60 aacggtcaaa ccaaagtgat cgaaaacgca
gaaggcgcac gcaccacgcc gtccgttatc 120 gcttatttgg acggcggcga
aatcctcgtc ggtgcgcctg ccaaacgcca agcggtaacc 180 aacgccaaaa
acaccattta cgccgccaaa cgtttgatcg gccacaaatt tgaagacaaa 240
gaagtccaac gcgacatcga atctatgcct ttcgaaatca tcaaagccaa caacggcgac
300 gcatgggtaa aagcacaagg caaagagctg tctcctcctc aaatttccgc
agaagtcctg 360 cgtaaaatga aagaagccgc cgaagcttac ttgggcgaaa
aagtaaccga agccgtgatt 420 accgtccctg cctacttcaa cgacagccaa
cgtcaagcca ccaaagacgc aggccgtatc 480 gccggtttgg acgtgaaacg
catcatcaac gagccgaccg cagccgcttt ggcattcggt 540 atggacaaag
gcgacaacaa agaccgcaaa gtagccgtat atgacttggg cggcggtact 600
ttcgatattt ccatcatcga aatcgccaac ctcgacggcg acaaacaatt cgaagtattg
660 gcaaccaacg gcgatacctt cttgggcggt gaagacttcg accaacgcct
catcgaccac 720 atcatcgccg agttcaaaaa agaacaaggc attgatttga
aacaagacgt gatggctcta 780 caacgcctga aagaagctgc cgaaaaagcc
aaaatcgaat tgtccagcgg ccagcaaacc 840 gaaattaacc tgccgtacat
caccatggac gcaaccggcc cgaaacactt ggcgatgaaa 900 attacccgcg
ccaaattcga aagcctggtt gaagacctga ttacccgctc tatcgaacct 960
tgcaaaattg cattgaaaga tgccggcttg agcaccggcg acatcgacga cgtaatcttg
1020 gtcggcgggc agtcccgtat gccgaaagta caagaagccg ttaaagcctt
cttcggcaaa 1080 gaaccgcgca aagacgtgaa ccctgacgaa gccgttgccg
taggcgcagc gatccaaggc 1140 gaagtattga gcggcggccg cagcgacgta
ttgctactgg acgtaactcc tctgtctttg 1200 ggtatcgaaa ccatgggcgg
cgtgatgacc aaactgattc agaagaacac caccatcccg 1260 accaaagcgt
cgcaagtgtt ctctaccgcc gaagacaacc aaagcgcagt aaccatccac 1320
gtactgcaag gcgaacgcga acgcgcttct gccaacaaat ctttgggtca gttcaacttg
1380 ggcgacatcg cacctgcacc gcgcggtatg ccgcaaatcg aagtaacctt
cgacatcgac 1440 gccaacggca tcctgcacgt ttccgccaaa gacaaaggca
ccggtaaagc agccaacatc 1500 accatccaag gttcttcagg tttgagcgaa
gaagaaatcg aacgcatggt gaaagatgcc 1560 gaagccaatg ccgaggaaga
taaaaaactg actgaattgg tcgcttcccg caaccaagcc 1620 gaagccctga
ttcactctgt gaaaaaatct ttggccgact acggcgacaa actcgatgca 1680
gccgagaaag aaaaaattga agccgcgctg aaagaagccg aagaagcagt taaaggcgac
1740 gacaaagccg ctatcgatgc caaaaccgag gcgctgggcg cagccagcca
aaaactgggg 1800 gaaatggttt acgctcaagc acaagctgaa gcccaagcag
gcgaaagcga acaagccaat 1860 gcttctgcaa agaaagacga tgatgtcgta
gatgccgact ttgaagaagt aaaagacgac 1920 aaaaaataa 1929 4 1989 DNA
Nesseria meningitidis 4 atgggcagca gccatcatca tcatcatcac agcagcggcc
tggtgccgcg cggcagccat 60 atggcaaaag taatcggtat cgacttaggt
acaaccaact cttgtttggc catttccgaa 120 aacggtcaaa ccaaagtgat
cgaaaacgca gaaggcgcac gcaccacgcc gtccgttatc 180 gcttatttgg
acggcggcga aatcctcgtc ggtgcgcctg ccaaacgcca agcggtaacc 240
aacgccaaaa acaccattta cgccgccaaa cgtttgatcg gccacaaatt tgaagacaaa
300 gaagtccaac gcgacatcga atctatgcct ttcgaaatca tcaaagccaa
caacggcgac 360 gcatgggtaa aagcacaagg caaagagctg tctcctcctc
aaatttccgc agaagtcctg 420 cgtaaaatga aagaagccgc cgaagcttac
ttgggcgaaa aagtaaccga agccgtgatt 480 accgtccctg cctacttcaa
cgacagccaa cgtcaagcca ccaaagacgc aggccgtatc 540 gccggtttgg
acgtgaaacg catcatcaac gagccgaccg cagccgcttt ggcattcggt 600
atggacaaag gcgacaacaa agaccgcaaa gtagccgtat atgacttggg cggcggtact
660 ttcgatattt ccatcatcga aatcgccaac ctcgacggcg acaaacaatt
cgaagtattg 720 gcaaccaacg gcgatacctt cttgggcggt gaagacttcg
accaacgcct catcgaccac 780 atcatcgccg agttcaaaaa agaacaaggc
attgatttga aacaagacgt gatggctcta 840 caacgcctga aagaagctgc
cgaaaaagcc aaaatcgaat tgtccagcgg ccagcaaacc 900 gaaattaacc
tgccgtacat caccatggac gcaaccggcc cgaaacactt ggcgatgaaa 960
attacccgcg ccaaattcga aagcctggtt gaagacctga ttacccgctc tatcgaacct
1020 tgcaaaattg cattgaaaga tgccggcttg agcaccggcg acatcgacga
cgtaatcttg 1080 gtcggcgggc agtcccgtat gccgaaagta caagaagccg
ttaaagcctt cttcggcaaa 1140 gaaccgcgca aagacgtgaa ccctgacgaa
gccgttgccg taggcgcagc gatccaaggc 1200 gaagtattga gcggcggccg
cagcgacgta ttgctactgg acgtaactcc tctgtctttg 1260 ggtatcgaaa
ccatgggcgg cgtgatgacc aaactgattc agaagaacac caccatcccg 1320
accaaagcgt cgcaagtgtt ctctaccgcc gaagacaacc aaagcgcagt aaccatccac
1380 gtactgcaag gcgaacgcga acgcgcttct gccaacaaat ctttgggtca
gttcaacttg 1440 ggcgacatcg cacctgcacc gcgcggtatg ccgcaaatcg
aagtaacctt cgacatcgac 1500 gccaacggca tcctgcacgt ttccgccaaa
gacaaaggca ccggtaaagc agccaacatc 1560 accatccaag gttcttcagg
tttgagcgaa gaagaaatcg aacgcatggt gaaagatgcc 1620 gaagccaatg
ccgaggaaga taaaaaactg actgaattgg tcgcttcccg caaccaagcc 1680
gaagccctga ttcactctgt gaaaaaatct ttggccgact acggcgacaa actcgatgca
1740 gccgagaaag aaaaaattga agccgcgctg aaagaagccg aagaagcagt
taaaggcgac 1800 gacaaagccg ctatcgatgc caaaaccgag gcgctgggcg
cagccagcca aaaactgggg 1860 gaaatggttt acgctcaagc acaagctgaa
gcccaagcag gcgaaagcga acaagccaat 1920 gcttctgcaa agaaagacga
tgatgtcgta gatgccgact ttgaagaagt aaaagacgac 1980 aaaaaataa 1989 5
2480 DNA Aspergillus fumigatus 5 gtacgaattt ccccttccga cgatccgaga
acgtccctcg ggaaggccac acgtgacctt 60 ctaggagctt ctcccgccaa
gacatccggg gatcgagaat cgcctggaaa aatttcgaga 120 ctttggcttc
atctccccag ctttcatctc cattccatct tccttacctt ctattccccc 180
tcttctcttc cttctctgca cctgttcttg ctctgggagg ttcgatcggg cagtagtgtt
240 catcttaacg ttgattatat tctcttctat cccgtccttt catcaccctt
ctttccataa 300 tgcagagagc tctttcttcc aggacgtctg tcctttccgc
tgcctccaaa cgggctgctt 360 tcaccaagcc cgctggcctt aacctgcagc
agcagcgttt cgcccacaag gtatgttttc 420 atctacaatc tagaatttta
agcttctgaa gtggtgccaa tttctccgtg tcacccggag 480 ctcaaccccg
ataccttgct aacgaacttt caggagctca agttcggtgt cgaagcccgt 540
gctcagctcc tcaagggtgt tgacactctg gccaaggccg tgacttcgac tcttggtcct
600 aagggtcgta acgtccttat cgagtctccc tatggctccc ctaagatcac
caagggtacg 660 tttgactcga gttaacccaa gtcgctgctt tcacaaacga
attgtggttc tgactaaaaa 720 tagatggtgt ctctgttgcc aaggccatca
ctctccaaga caagttcgag aacctcggtg 780 ctcgcctcct ccaggatgtc
gcttctaaga ccaacgagat tgctggtgac ggtaccacca 840 ccgctaccgt
ccttgcccgt gccatcttct ctgagaccgt gaagaatgtt gctgctggct 900
gcaaccccat ggatctgcgc cgcggtatcc aggctgctgt tgatgctgtc gtcgactacc
960 tccagaagaa caagcgtgac atcaccaccg gtgaggagat cgctcaggtt
gctactatct 1020 ccgctaacgg tgacacccac attggtaagc tgatctccac
cgccatggag cgtgttggca 1080 aggagggtgt catcactgtc aaggagggca
agaccattga ggatgagctc gaggtcactg 1140 agggtatgcg cttcgaccgt
ggatacacct ccccctactt catcaccgat accaagtccc 1200 agaaggttga
gttcgagaag cctctgattc tgctgtctga gaagaagatc tctgccgttc 1260
aggacatcat ccccgccctt gaggcctcca ccaccctccg ccgccccctg gttattatcg
1320 cagaggacat tgagggtgag gctctcgccg tctgcattct gaacaagctt
cgtggccagc 1380 tgcaggtcgc tgctgtcaag gctcctggat tcggtgacaa
ccgcaagagc atcctgggcg 1440 atcttgccgt ccttaccaac ggtaccgtct
tcactgatga gctcgacatc aaactcgaga 1500 agcttacccc cgatatgctt
ggttccaccg gcgccatcac catcaccaag gaggacacca 1560 tcatcctgaa
cggggagggc agcaaggacg ccattgccca gcgctgcgag cagattcgcg 1620
gtgtcatggc ggaccccagc acctccgaat acgagaagga gaagctccag gagcgtctag
1680 ctaagctctc tggcggtgtt gccgtcatca aggtcggtgg tgcctccgag
gttgaggtcg 1740 gtgagaagaa ggaccgtgtt gtcgatgctc tcaatgctac
ccgtgctgct gttgaggagg 1800 gtatcctccc cggtggtggt accgcccttc
tcaaggccgc cgccaacggc cttgacaatg 1860 tcaagcccga gaacttcgac
cagcaactcg gtgtgagcat catcaagaat gccatcaccc 1920 gccccgctcg
caccattgtt gagaacgccg gcctcgaggg cagcgtcatt gtcggcaagc 1980
tgaccgacga gttcgccaag gacttcaacc gcggtttcga cagctccaag ggcgagtacg
2040 tcgacatgat ctccagcggt atcctcgatc ccctcaaggt tgttcgcacc
gctctgctcg 2100 acgccagcgg tgtcgcctcc ctgctcggta ccactgaggt
cgctattgtt gaggcccctg 2160 aggagaaggg ccccgctgct cctggcatgg
gtggtatggg tggtatgggc ggcatgggtg 2220 gcggcatgtt ctaagctgct
cccagttgcc tttgctacca tagcctcttc catgatttaa 2280 aggtttaact
tccctttcga gcgtgtcttt gcatgtacga gcatttcctg atatatcggt 2340
gttgagagtt ttctgtaatt tttcctttgt ttctgatgtg ttacacgcct tgacagcccc
2400 ttcacctact ccgacttcgt cttatacctc gatactcata tctccctctt
cgacccgcct 2460 cccctttgat tgactcgatc 2480 6 1653 DNA Aspergillus
fumigatus 6 atgaaagagc tcaagttcgg tgtcgaagcc cgtgctcagc tcctcaaggg
tgttgacact 60 ctggccaagg ccgtgacttc gactcttggt cctaagggtc
gtaacgtcct tatcgagtct 120 ccctatggct cccctaagat caccaaggat
ggtgtctctg ttgccaaggc catcactctc 180 caagacaagt tcgagaacct
cggtgctcgc ctcctccagg atgtcgcttc taagaccaac 240 gagattgctg
gtgacggtac caccaccgct accgtccttg cccgtgccat cttctctgag 300
accgtgaaga atgttgctgc tggctgcaac cccatggatc tgcgccgcgg tatccaggct
360 gctgttgatg ctgtcgtcga ctacctccag aagaacaagc gtgacatcac
caccggtgag 420 gagatcgctc aggttgctac tatctccgct aacggtgaca
cccacattgg taagctgatc 480 tccaccgcca tggagcgtgt tggcaaggag
ggtgtcatca ctgtcaagga gggcaagacc 540 attgaggatg agctcgaggt
cactgagggt atgcgcttcg accgtggata cacctccccc 600 tacttcatca
ccgataccaa gtcccagaag gttgagttcg agaagcctct gattctgctg 660
tctgagaaga agatctctgc cgttcaggac atcatccccg cccttgaggc ctccaccacc
720 ctccgccgcc ccctggttat tatcgcagag gacattgagg gtgaggctct
cgccgtctgc 780 attctgaaca agcttcgtgg ccagctgcag gtcgctgctg
tcaaggctcc tggattcggt 840 gacaaccgca agagcatcct gggcgatctt
gccgtcctta ccaacggtac cgtcttcact 900 gatgagctcg acatcaaact
cgagaagctt acccccgata tgcttggttc caccggcgcc 960 atcaccatca
ccaaggagga caccatcatc ctgaacgggg agggcagcaa ggacgccatt 1020
gcccagcgct gcgagcagat tcgcggtgtc atggcggacc ccagcacctc cgaatacgag
1080 aaggagaagc tccaggagcg tctagctaag ctctctggcg gtgttgccgt
catcaaggtc 1140 ggtggtgcct ccgaggttga ggtcggtgag aagaaggacc
gtgttgtcga tgctctcaat 1200 gctacccgtg ctgctgttga ggagggtatc
ctccccggtg gtggtaccgc ccttctcaag 1260 gccgccgcca acggccttga
caatgtcaag cccgagaact tcgaccagca actcggtgtg 1320 agcatcatca
agaatgccat cacccgcccc gctcgcacca ttgttgagaa cgccggcctc 1380
gagggcagcg tcattgtcgg caagctgacc gacgagttcg ccaaggactt caaccgcggt
1440 ttcgacagct ccaagggcga gtacgtcgac atgatctcca gcggtatcct
cgatcccctc 1500 aaggttgttc gcaccgctct gctcgacgcc agcggtgtcg
cctccctgct cggtaccact 1560 gaggtcgcta ttgttgaggc ccctgaggag
aagggccccg ctgctcctgg catgggtggt 1620 atgggtggta tgggcggcat
gggcggcatg tag 1653 7 1713 DNA Aspergillus fumigatus 7 atgggcagca
gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atgaaagagc tcaagttcgg tgtcgaagcc cgtgctcagc tcctcaaggg tgttgacact
120 ctggccaagg ccgtgacttc gactcttggt cctaagggtc gtaacgtcct
tatcgagtct 180 ccctatggct cccctaagat caccaaggat ggtgtctctg
ttgccaaggc catcactctc 240 caagacaagt tcgagaacct cggtgctcgc
ctcctccagg atgtcgcttc taagaccaac 300 gagattgctg gtgacggtac
caccaccgct accgtccttg cccgtgccat cttctctgag 360 accgtgaaga
atgttgctgc tggctgcaac cccatggatc tgcgccgcgg tatccaggct 420
gctgttgatg ctgtcgtcga ctacctccag aagaacaagc gtgacatcac caccggtgag
480 gagatcgctc aggttgctac tatctccgct aacggtgaca cccacattgg
taagctgatc 540 tccaccgcca tggagcgtgt tggcaaggag ggtgtcatca
ctgtcaagga gggcaagacc 600 attgaggatg agctcgaggt cactgagggt
atgcgcttcg accgtggata cacctccccc 660 tacttcatca ccgataccaa
gtcccagaag gttgagttcg agaagcctct gattctgctg 720 tctgagaaga
agatctctgc cgttcaggac atcatccccg cccttgaggc ctccaccacc 780
ctccgccgcc ccctggttat tatcgcagag gacattgagg gtgaggctct cgccgtctgc
840 attctgaaca agcttcgtgg ccagctgcag gtcgctgctg tcaaggctcc
tggattcggt 900 gacaaccgca agagcatcct gggcgatctt gccgtcctta
ccaacggtac cgtcttcact 960 gatgagctcg acatcaaact cgagaagctt
acccccgata tgcttggttc caccggcgcc 1020 atcaccatca ccaaggagga
caccatcatc ctgaacgggg agggcagcaa ggacgccatt 1080 gcccagcgct
gcgagcagat tcgcggtgtc atggcggacc ccagcacctc cgaatacgag 1140
aaggagaagc tccaggagcg tctagctaag ctctctggcg gtgttgccgt catcaaggtc
1200 ggtggtgcct ccgaggttga ggtcggtgag aagaaggacc gtgttgtcga
tgctctcaat 1260 gctacccgtg ctgctgttga ggagggtatc ctccccggtg
gtggtaccgc ccttctcaag 1320 gccgccgcca acggccttga caatgtcaag
cccgagaact tcgaccagca actcggtgtg 1380 agcatcatca agaatgccat
cacccgcccc gctcgcacca ttgttgagaa cgccggcctc 1440 gagggcagcg
tcattgtcgg caagctgacc gacgagttcg ccaaggactt caaccgcggt 1500
ttcgacagct ccaagggcga gtacgtcgac atgatctcca gcggtatcct cgatcccctc
1560 aaggttgttc gcaccgctct gctcgacgcc agcggtgtcg cctccctgct
cggtaccact 1620 gaggtcgcta ttgttgaggc ccctgaggag aagggccccg
ctgctcctgg catgggtggt 1680 atgggtggta tgggcggcat gggcggcatg tag
1713 8 2051 DNA Candida glabrata 8 ccgggtaaag tacctgattg cgcacttaca
gctaacagct gacgcactcg agaaatctgc 60 ccgttttgtt catggaaact
tgaagaaaat cagggaaatc gttactgcgc tctctctaac 120 gcttgcaagc
tcctggaata caaattcgca aagtatataa ctctatagct ttcaaccttg 180
ttactgtgga gtagctgtta agggatagag acataagata aaccatacca tacataaatc
240 accccccata taaacaaatg ttgagagctg ttgcacgttc gcaggttaga
tctttgagaa 300 acgctcgttt gtactccagt ttcaaggagt tgaagttcgg
tgtcgaaggc agagctgctc 360
tgcttcgtgg tgtcgagact ttggccgacg ctgtctctgc cacgctgggg cctaagggta
420 gaaatgtgct gatcgagcag ccattcggag caccaaagat caccaaggat
ggtgtcaccg 480 tggccagatc cattactttg gaggacaagt tcgagaacat
gggtgctaag cttctgcaag 540 aagttgcctc caagactaac gaggccgccg
gtgacggtac cacctccgcc actgtcttgg 600 gtagagccat cttcaccgag
tccgtcaaga acgtcgctgc cggttgcaac cctatggatt 660 tgagaagggg
ttcccaggcc gccgtcgaga aggtcatcca attcttgact gaaaacaaga 720
aggagatcac cacttctgag gaaatcgccc aggtggccac catctcagct aacggtgacg
780 ctcacgtcgg taagttgctt gcctccgcca tggaaaaggt tggcaaggaa
ggtgttatca 840 ccatcagaga aggcagaact ttggaagacg aactagaagt
caccgaaggt atgagatttg 900 accgtggttt catctcccca tacttcatca
ctgacgcaaa gtccggcaag gtagaattcg 960 aaaagccatt gttgttgttg
agcgaaaaga agatctcttc catccaagac atcttgccag 1020 ctttggaact
atctaaccaa agtagaagac cactattgat catcgccgaa gatgtcgacg 1080
gtgaagccct agctgcttgt atcctaaaca agttgagagg ccaagtcaag gtctgtgccg
1140 ttaaggctcc aggtttcggt gacaacagaa agaacattct aggtgatgtc
gccatcttga 1200 ccggcagtac tgtttttact gaagaattgg acttgaagcc
agaacaagcc actatggaac 1260 acctaggttc ctgtgactcc attactatca
caaaggaaga cactgttatc ctaaacggta 1320 acggctccaa ggactctatc
caagaaagaa ttgaacagat caagaactcc attgatgtca 1380 ccactactaa
ctcttacgag aaggagaagc tacaagaaag acttgccaag ttatccggtg 1440
gtgttgctgt catcagggtt ggtggtgctt ctgaagttga agtcggtgaa aagaaggacc
1500 gttacgatga cgctttgaat gccaccagag ctgccgttga agaaggtatc
ttaccaggtg 1560 gtggtactgc tttggttaag gcctctagag ttttagacga
agtcaagact gagaacttcg 1620 accaaaaatt gggagttgac attatcagaa
aggccattac tagaccagct aagcaaatta 1680 ttgagaacgc cggtgaagaa
ggctccgtta ttgtcggtaa gcttgttgat gaatttggcg 1740 aagattttgc
taagggttac gactccgcta agggagaatt cactgatatg ttggctgccg 1800
gtattattga cccattcaaa gtcgttagat ctggtctggt cgacgcttcc ggtgttgctt
1860 ccttgttggc tactaccgaa gttgccatcg ttgacgctcc tgaaccagct
ccagctgctg 1920 gtgccccagg tggtggtatg ccaggtatgc caggtatgat
gtaaaaggtc taacttttgc 1980 aatcatgctg gtgaaaatga agcaaatcat
tacatagagt ggtaaaatct tcaagaccaa 2040 atagcttgta c 2051 9 1647 DNA
Candida glabrata 9 atggccaagg agttgaagtt tggggtcgaa ggcagagctg
ctctgcttcg tggtgtcgag 60 actttggccg acgctgtctc tgccacgctg
gggcctaagg gtagaaatgt gctgatcgag 120 cagccattcg gagcaccaaa
gatcaccaag gatggtgtca ccgtggccag atccattact 180 ttggaggaca
agttcgagaa catgggtgct aagcttctgc aagaagttgc ctccaagact 240
aacgaggccg ccggtgacgg taccacctcc gccactgtct tgggtagagc catcttcacc
300 gagtccgtca agaacgtcgc tgccggttgc aaccctatgg atttgagaag
gggttcccag 360 gccgccgtcg agaaggtcat ccaattcttg actgaaaaca
agaaggagat caccacttct 420 gaggaaatcg cccaggtggc caccatctca
gctaacggtg acgctcacgt cggtaagttg 480 cttgcctccg ccatggaaaa
ggttggcaag gaaggtgtta tcaccatcag agaaggcaga 540 actttggaag
acgaactaga agtcaccgaa ggtatgagat ttgaccgtgg tttcatctcc 600
ccatacttca tcactgacgc aaagtccggc aaggtagaat tcgaaaagcc attgttgttg
660 ttgagcgaaa agaagatctc ttccatccaa gacatcttgc cagctttgga
actatctaac 720 caaagtagaa gaccactatt gatcatcgcc gaagatgtcg
acggtgaagc cctagctgct 780 tgtatcctaa acaagttgag aggccaagtc
aaggtctgtg ccgttaaggc tccaggtttc 840 ggtgacaaca gaaagaacat
tctaggtgat gtcgccatct tgaccggcag tactgttttt 900 actgaagaat
tggacttgaa gccagaacaa gccactatgg aacacctagg ttcctgtgac 960
tccattacta tcacaaagga agacactgtt atcctaaacg gtaacggctc caaggactct
1020 atccaagaaa gaattgaaca gatcaagaac tccattgatg tcaccactac
taactcttac 1080 gagaaggaga agctacaaga aagacttgcc aagttatccg
gtggtgttgc tgtcatcagg 1140 gttggtggtg cttctgaagt tgaagtcggt
gaaaagaagg accgttacga tgacgctttg 1200 aatgccacca gagctgccgt
tgaagaaggt atcttaccag gtggtggtac tgctttggtt 1260 aaggcctcta
gagttttaga cgaagtcaag actgagaact tcgaccaaaa attgggagtt 1320
gacattatca gaaaggccat tactagacca gctaagcaaa ttattgagaa cgccggtgaa
1380 gaaggctccg ttattgtcgg taagcttgtt gatgaatttg gcgaagattt
tgctaagggt 1440 tacgactccg ctaagggaga attcactgat atgttggctg
ccggtattat tgacccattc 1500 aaagtcgtta gatctggtct ggtcgacgct
tccggtgttg cttccttgtt ggctactacc 1560 gaagttgcca tcgttgacgc
tcctgaacca gctccagctg ctggtgcccc aggtggtggt 1620 atgccaggta
tgccaggtat gatgtaa 1647 10 1707 DNA Candida glabrata 10 atgggcagca
gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggccaagg agttgaagtt tggggtcgaa ggcagagctg ctctgcttcg tggtgtcgag
120 actttggccg acgctgtctc tgccacgctg gggcctaagg gtagaaatgt
gctgatcgag 180 cagccattcg gagcaccaaa gatcaccaag gatggtgtca
ccgtggccag atccattact 240 ttggaggaca agttcgagaa catgggtgct
aagcttctgc aagaagttgc ctccaagact 300 aacgaggccg ccggtgacgg
taccacctcc gccactgtct tgggtagagc catcttcacc 360 gagtccgtca
agaacgtcgc tgccggttgc aaccctatgg atttgagaag gggttcccag 420
gccgccgtcg agaaggtcat ccaattcttg actgaaaaca agaaggagat caccacttct
480 gaggaaatcg cccaggtggc caccatctca gctaacggtg acgctcacgt
cggtaagttg 540 cttgcctccg ccatggaaaa ggttggcaag gaaggtgtta
tcaccatcag agaaggcaga 600 actttggaag acgaactaga agtcaccgaa
ggtatgagat ttgaccgtgg tttcatctcc 660 ccatacttca tcactgacgc
aaagtccggc aaggtagaat tcgaaaagcc attgttgttg 720 ttgagcgaaa
agaagatctc ttccatccaa gacatcttgc cagctttgga actatctaac 780
caaagtagaa gaccactatt gatcatcgcc gaagatgtcg acggtgaagc cctagctgct
840 tgtatcctaa acaagttgag aggccaagtc aaggtctgtg ccgttaaggc
tccaggtttc 900 ggtgacaaca gaaagaacat tctaggtgat gtcgccatct
tgaccggcag tactgttttt 960 actgaagaat tggacttgaa gccagaacaa
gccactatgg aacacctagg ttcctgtgac 1020 tccattacta tcacaaagga
agacactgtt atcctaaacg gtaacggctc caaggactct 1080 atccaagaaa
gaattgaaca gatcaagaac tccattgatg tcaccactac taactcttac 1140
gagaaggaga agctacaaga aagacttgcc aagttatccg gtggtgttgc tgtcatcagg
1200 gttggtggtg cttctgaagt tgaagtcggt gaaaagaagg accgttacga
tgacgctttg 1260 aatgccacca gagctgccgt tgaagaaggt atcttaccag
gtggtggtac tgctttggtt 1320 aaggcctcta gagttttaga cgaagtcaag
actgagaact tcgaccaaaa attgggagtt 1380 gacattatca gaaaggccat
tactagacca gctaagcaaa ttattgagaa cgccggtgaa 1440 gaaggctccg
ttattgtcgg taagcttgtt gatgaatttg gcgaagattt tgctaagggt 1500
tacgactccg ctaagggaga attcactgat atgttggctg ccggtattat tgacccattc
1560 aaagtcgtta gatctggtct ggtcgacgct tccggtgttg cttccttgtt
ggctactacc 1620 gaagttgcca tcgttgacgc tcctgaacca gctccagctg
ctggtgcccc aggtggtggt 1680 atgccaggta tgccaggtat gatgtaa 1707 11
1005 DNA Neisseria meningitidis 11 cctgcdtatt tcaacgacag ccaacgtcaa
gccaccaaag acgcaggccg tatcgccggt 60 ttggacgtga aacgcatcat
caacgagccg accgcagccg ctttggcatt cggtatggac 120 aaaggcgaca
acaaagaccg caaagtagcc gtatatgact tgggcggcgg tactttcgat 180
atttccatca tcgaaatcgc caacctcgac ggcgacaaac aattcgaagt attggcaacc
240 aacggcgata ccttcttggg cggtgaagac ttcgaccaac gcctcatcga
ccacatcatc 300 gccgagttca aaaaagaaca aggcattgat ttgaaacaag
acgtgatggc tctacaacgc 360 ctgaaagaag ctgccgaaaa agccaaaatc
gaattgtcca gcggccagca aaccgaaatt 420 aacctgccgt acatcaccat
ggacgcaacc ggcccgaaac acttggcgat gaaaattacc 480 cgcgccaaat
tcgaaagcct ggttgaagac ctgattaccc gctctatcga accttgcaaa 540
attgcattga aagatgccgg cttgagcacc ggcgacatcg acgacgtaat cttggtcggc
600 gggcagtccc gtatgccgaa agtacaagaa gccgttaaag ccttcttcgg
caaagaaccg 660 cgcaaagacg tgaaccctga cgaagccgtt gccgtaggcg
cagcgatcca aggcgaagta 720 ttgagcggcg gccgcagcga cgtattgcta
ctggacgtaa ctcctctgtc tttgggtatc 780 gaaaccatgg gcggcgtgat
gaccaaactg attcagaaga acaccaccat cccgaccaaa 840 gcgtcgcaag
tgttctctac cgccgaagac aaccaaagcg cagtaaccat ccacgtactg 900
caaggcgaac gcgaacgcgc ttctgccaac aaatctttgg gtcagttcaa cttgggcgac
960 atcgcacctg caccgcgcgg tatgccacaa atcgaagtaa chttt 1005 12 415
PRT Neisseria meningitidis 12 Met Ala Lys Val Ile Gly Ile Asp Leu
Gly Thr Thr Asn Ser Cys Leu 1 5 10 15 Ala Ile Ser Glu Asn Gly Gln
Thr Lys Val Ile Glu Asn Ala Glu Gly 20 25 30 Ala Arg Thr Thr Pro
Ser Val Ile Ala Tyr Leu Asp Gly Gly Glu Ile 35 40 45 Leu Val Gly
Ala Pro Ala Lys Arg Gln Ala Val Thr Asn Ala Lys Asn 50 55 60 Thr
Ile Tyr Ala Ala Lys Arg Leu Ile Gly His Lys Phe Glu Asp Lys 65 70
75 80 Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp Ala
Gly 85 90 95 Arg Ile Ala Gly Leu Asp Val Lys Arg Ile Ile Asn Glu
Pro Thr Ala 100 105 110 Ala Ala Leu Ala Phe Gly Met Asp Lys Gly Asp
Asn Lys Asp Arg Lys 115 120 125 Val Ala Val Tyr Asp Leu Gly Gly Gly
Thr Phe Asp Ile Ser Ile Ile 130 135 140 Glu Ile Ala Asn Leu Asp Gly
Asp Lys Gln Phe Glu Val Leu Ala Thr 145 150 155 160 Asn Gly Asp Thr
Phe Leu Gly Gly Glu Asp Phe Asp Gln Arg Leu Ile 165 170 175 Asp His
Ile Ile Ala Glu Phe Lys Lys Glu Gln Gly Ile Asp Leu Lys 180 185 190
Gln Asp Val Met Ala Leu Gln Arg Leu Lys Glu Ala Ala Glu Lys Ala 195
200 205 Lys Ile Glu Leu Ser Ser Gly Gln Gln Thr Glu Ile Asn Leu Pro
Tyr 210 215 220 Ile Thr Met Asp Ala Thr Gly Pro Lys His Leu Ala Met
Lys Ile Thr 225 230 235 240 Arg Ala Lys Phe Glu Ser Leu Val Glu Asp
Leu Ile Thr Arg Ser Ile 245 250 255 Glu Pro Cys Lys Ile Ala Leu Lys
Asp Ala Gly Leu Ser Thr Gly Asp 260 265 270 Ile Asp Asp Val Ile Leu
Val Gly Gly Gln Ser Arg Met Pro Lys Val 275 280 285 Gln Glu Ala Val
Lys Ala Phe Phe Gly Lys Glu Pro Arg Lys Asp Val 290 295 300 Asn Pro
Asp Glu Ala Val Ala Val Gly Ala Ala Ile Gln Gly Glu Val 305 310 315
320 Leu Ser Gly Gly Arg Ser Asp Val Leu Leu Leu Asp Val Thr Pro Leu
325 330 335 Ser Leu Gly Ile Glu Thr Met Gly Gly Val Met Thr Lys Leu
Ile Gln 340 345 350 Lys Asn Thr Thr Ile Pro Thr Lys Ala Ser Gln Val
Phe Ser Thr Ala 355 360 365 Glu Asp Asn Gln Ser Ala Val Thr Ile His
Val Leu Gln Gly Glu Arg 370 375 380 Glu Arg Ala Ser Ala Asn Lys Ser
Leu Gly Gln Phe Asn Leu Gly Asp 385 390 395 400 Ile Ala Pro Ala Pro
Arg Gly Met Pro Gln Ile Glu Val Thr Phe 405 410 415 13 642 PRT
Neisseria meningitidis 13 Met Ala Lys Val Ile Gly Ile Asp Leu Gly
Thr Thr Asn Ser Cys Leu 1 5 10 15 Ala Ile Ser Glu Asn Gly Gln Thr
Lys Val Ile Glu Asn Ala Glu Gly 20 25 30 Ala Arg Thr Thr Pro Ser
Val Ile Ala Tyr Leu Asp Gly Gly Glu Ile 35 40 45 Leu Val Gly Ala
Pro Ala Lys Arg Gln Ala Val Thr Asn Ala Lys Asn 50 55 60 Thr Ile
Tyr Ala Ala Lys Arg Leu Ile Gly His Lys Phe Glu Asp Lys 65 70 75 80
Glu Val Gln Arg Asp Ile Glu Ser Met Pro Phe Glu Ile Ile Lys Ala 85
90 95 Asn Asn Gly Asp Ala Trp Val Lys Ala Gln Gly Lys Glu Leu Ser
Pro 100 105 110 Pro Gln Ile Ser Ala Glu Val Leu Arg Lys Met Lys Glu
Ala Ala Glu 115 120 125 Ala Tyr Leu Gly Glu Lys Val Thr Glu Ala Val
Ile Thr Val Pro Ala 130 135 140 Tyr Phe Asn Asp Ser Gln Arg Gln Ala
Thr Lys Asp Ala Gly Arg Ile 145 150 155 160 Ala Gly Leu Asp Val Lys
Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala 165 170 175 Leu Ala Phe Gly
Met Asp Lys Gly Asp Asn Lys Asp Arg Lys Val Ala 180 185 190 Val Tyr
Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile Ile Glu Ile 195 200 205
Ala Asn Leu Asp Gly Asp Lys Gln Phe Glu Val Leu Ala Thr Asn Gly 210
215 220 Asp Thr Phe Leu Gly Gly Glu Asp Phe Asp Gln Arg Leu Ile Asp
His 225 230 235 240 Ile Ile Ala Glu Phe Lys Lys Glu Gln Gly Ile Asp
Leu Lys Gln Asp 245 250 255 Val Met Ala Leu Gln Arg Leu Lys Glu Ala
Ala Glu Lys Ala Lys Ile 260 265 270 Glu Leu Ser Ser Gly Gln Gln Thr
Glu Ile Asn Leu Pro Tyr Ile Thr 275 280 285 Met Asp Ala Thr Gly Pro
Lys His Leu Ala Met Lys Ile Thr Arg Ala 290 295 300 Lys Phe Glu Ser
Leu Val Glu Asp Leu Ile Thr Arg Ser Ile Glu Pro 305 310 315 320 Cys
Lys Ile Ala Leu Lys Asp Ala Gly Leu Ser Thr Gly Asp Ile Asp 325 330
335 Asp Val Ile Leu Val Gly Gly Gln Ser Arg Met Pro Lys Val Gln Glu
340 345 350 Ala Val Lys Ala Phe Phe Gly Lys Glu Pro Arg Lys Asp Val
Asn Pro 355 360 365 Asp Glu Ala Val Ala Val Gly Ala Ala Ile Gln Gly
Glu Val Leu Ser 370 375 380 Gly Gly Arg Ser Asp Val Leu Leu Leu Asp
Val Thr Pro Leu Ser Leu 385 390 395 400 Gly Ile Glu Thr Met Gly Gly
Val Met Thr Lys Leu Ile Gln Lys Asn 405 410 415 Thr Thr Ile Pro Thr
Lys Ala Ser Gln Val Phe Ser Thr Ala Glu Asp 420 425 430 Asn Gln Ser
Ala Val Thr Ile His Val Leu Gln Gly Glu Arg Glu Arg 435 440 445 Ala
Ser Ala Asn Lys Ser Leu Gly Gln Phe Asn Leu Gly Asp Ile Ala 450 455
460 Pro Ala Pro Arg Gly Met Pro Gln Ile Glu Val Thr Phe Asp Ile Asp
465 470 475 480 Ala Asn Gly Ile Leu His Val Ser Ala Lys Asp Lys Gly
Thr Gly Lys 485 490 495 Ala Ala Asn Ile Thr Ile Gln Gly Ser Ser Gly
Leu Gly Glu Glu Glu 500 505 510 Ile Glu Arg Met Val Lys Asp Ala Glu
Ala Asn Ala Glu Glu Asp Lys 515 520 525 Lys Leu Thr Glu Leu Val Ala
Ser Arg Asn Gln Ala Glu Ala Leu Ile 530 535 540 His Ser Val Lys Lys
Ser Leu Ala Asp Tyr Gly Asp Lys Leu Asp Ala 545 550 555 560 Ala Glu
Lys Glu Lys Ile Glu Ala Ala Leu Lys Glu Ala Glu Glu Ala 565 570 575
Val Lys Gly Asp Asp Lys Ala Ala Ile Asp Ala Lys Thr Glu Ala Leu 580
585 590 Gly Ala Ala Ser Gln Lys Leu Gly Glu Met Val Tyr Ala Gln Ala
Gln 595 600 605 Ala Glu Ala Gln Ala Gly Glu Ser Glu Gln Ala Asn Ala
Ser Ala Lys 610 615 620 Lys Asp Asp Asp Val Val Asp Ala Asp Phe Glu
Glu Val Lys Asp Asp 625 630 635 640 Lys Lys 14 562 PRT Neisseria
meningitidis 14 Glu Val Gln Arg Asp Ile Glu Ser Met Pro Phe Glu Ile
Ile Lys Ala 1 5 10 15 Asn Asn Gly Asp Ala Trp Val Lys Ala Gln Gly
Lys Glu Leu Ser Pro 20 25 30 Pro Gln Ile Ser Ala Glu Val Leu Arg
Lys Met Lys Glu Ala Ala Glu 35 40 45 Ala Tyr Leu Gly Glu Lys Val
Thr Glu Ala Val Ile Thr Val Pro Ala 50 55 60 Tyr Phe Asn Asp Ser
Gln Arg Gln Ala Thr Lys Asp Ala Gly Arg Ile 65 70 75 80 Ala Gly Leu
Asp Val Lys Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala 85 90 95 Leu
Ala Phe Gly Met Asp Lys Gly Asp Asn Lys Asp Arg Lys Val Ala 100 105
110 Val Tyr Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile Ile Glu Ile
115 120 125 Ala Asn Leu Asp Gly Asp Lys Gln Phe Glu Val Leu Ala Thr
Asn Gly 130 135 140 Asp Thr Phe Leu Gly Gly Glu Asp Phe Asp Gln Arg
Leu Ile Asp His 145 150 155 160 Ile Ile Ala Glu Phe Lys Lys Glu Gln
Gly Ile Asp Leu Lys Gln Asp 165 170 175 Val Met Ala Leu Gln Arg Leu
Lys Glu Ala Ala Glu Lys Ala Lys Ile 180 185 190 Glu Leu Ser Ser Gly
Gln Gln Thr Glu Ile Asn Leu Pro Tyr Ile Thr 195 200 205 Met Asp Ala
Thr Gly Pro Lys His Leu Ala Met Lys Ile Thr Arg Ala 210 215 220 Lys
Phe Glu Ser Leu Val Glu Asp Leu Ile Thr Arg Ser Ile Glu Pro 225 230
235 240 Cys Lys Ile Ala Leu Lys Asp Ala Gly Leu Ser Thr Gly Asp Ile
Asp 245 250 255 Asp Val Ile Leu Val Gly Gly Gln Ser Arg Met Pro Lys
Val Gln Glu 260 265 270 Ala Val Lys Ala Phe Phe Gly Lys Glu Pro Arg
Lys Asp Val Asn Pro 275 280 285 Asp Glu Ala Val Ala Val Gly Ala Ala
Ile Gln Gly Glu Val Leu Ser 290 295 300 Gly Gly Arg Ser Asp Val Leu
Leu Leu Asp Val Thr Pro Leu Ser Leu 305 310 315 320 Gly Ile Glu Thr
Met Gly Gly Val Met Thr Lys Leu Ile Gln Lys Asn 325 330 335 Thr Thr
Ile Pro Thr Lys Ala Ser Gln Val Phe Ser Thr Ala Glu Asp 340 345 350
Asn Gln Ser Ala Val Thr Ile His Val Leu Gln
Gly Glu Arg Glu Arg 355 360 365 Ala Ser Ala Asn Lys Ser Leu Gly Gln
Phe Asn Leu Gly Asp Ile Ala 370 375 380 Pro Ala Pro Arg Gly Met Pro
Gln Ile Glu Val Thr Phe Asp Ile Asp 385 390 395 400 Ala Asn Gly Ile
Leu His Val Ser Ala Lys Asp Lys Gly Thr Gly Lys 405 410 415 Ala Ala
Asn Ile Thr Ile Gln Gly Ser Ser Gly Leu Ser Glu Glu Glu 420 425 430
Ile Glu Arg Met Val Lys Asp Ala Glu Ala Asn Ala Glu Glu Asp Lys 435
440 445 Lys Leu Thr Glu Leu Val Ala Ser Arg Asn Gln Ala Glu Ala Leu
Ile 450 455 460 His Ser Val Lys Lys Ser Leu Ala Asp Tyr Gly Asp Lys
Leu Asp Ala 465 470 475 480 Ala Glu Lys Glu Lys Ile Glu Ala Ala Leu
Lys Glu Ala Glu Glu Ala 485 490 495 Val Lys Gly Asp Asp Lys Ala Ala
Ile Asp Ala Lys Thr Glu Ala Leu 500 505 510 Gly Ala Ala Ser Gln Lys
Leu Gly Glu Met Val Tyr Ala Gln Ala Gln 515 520 525 Ala Glu Ala Gln
Ala Gly Glu Ser Glu Gln Ala Asn Ala Ser Ala Lys 530 535 540 Lys Asp
Asp Asp Val Val Asp Ala Asp Phe Glu Glu Val Lys Asp Asp 545 550 555
560 Lys Lys 15 642 PRT Neisseria meningitidis 15 Met Ala Lys Val
Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser Cys Leu 1 5 10 15 Ala Ile
Ser Glu Asn Gly Gln Thr Lys Val Ile Glu Asn Ala Glu Gly 20 25 30
Ala Arg Thr Thr Pro Ser Val Ile Ala Tyr Leu Asp Gly Gly Glu Ile 35
40 45 Leu Val Gly Ala Pro Ala Lys Arg Gln Ala Val Thr Asn Ala Lys
Asn 50 55 60 Thr Ile Tyr Ala Ala Lys Arg Leu Ile Gly His Lys Phe
Glu Asp Lys 65 70 75 80 Glu Val Gln Arg Asp Ile Glu Ser Met Pro Phe
Glu Ile Ile Lys Ala 85 90 95 Asn Asn Gly Asp Ala Trp Val Lys Ala
Gln Gly Lys Glu Leu Ser Pro 100 105 110 Pro Gln Ile Ser Ala Glu Val
Leu Arg Lys Met Lys Glu Ala Ala Glu 115 120 125 Ala Tyr Leu Gly Glu
Lys Val Thr Glu Ala Val Ile Thr Val Pro Ala 130 135 140 Tyr Phe Asn
Asp Ser Gln Arg Gln Ala Thr Lys Asp Ala Gly Arg Ile 145 150 155 160
Ala Gly Leu Asp Val Lys Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala 165
170 175 Leu Ala Phe Gly Met Asp Lys Gly Asp Asn Lys Asp Arg Lys Val
Ala 180 185 190 Val Tyr Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile
Ile Glu Ile 195 200 205 Ala Asn Leu Asp Gly Asp Lys Gln Phe Glu Val
Leu Ala Thr Asn Gly 210 215 220 Asp Thr Phe Leu Gly Gly Glu Asp Phe
Asp Gln Arg Leu Ile Asp His 225 230 235 240 Ile Ile Ala Glu Phe Lys
Lys Glu Gln Gly Ile Asp Leu Lys Gln Asp 245 250 255 Val Met Ala Leu
Gln Arg Leu Lys Glu Ala Ala Glu Lys Ala Lys Ile 260 265 270 Glu Leu
Ser Ser Gly Gln Gln Thr Glu Ile Asn Leu Pro Tyr Ile Thr 275 280 285
Met Asp Ala Thr Gly Pro Lys His Leu Ala Met Lys Ile Thr Arg Ala 290
295 300 Lys Phe Glu Ser Leu Val Glu Asp Leu Ile Thr Arg Ser Ile Glu
Pro 305 310 315 320 Cys Lys Ile Ala Leu Lys Asp Ala Gly Leu Ser Thr
Gly Asp Ile Asp 325 330 335 Asp Val Ile Leu Val Gly Gly Gln Ser Arg
Met Pro Lys Val Gln Glu 340 345 350 Ala Val Lys Ala Phe Phe Gly Lys
Glu Pro Arg Lys Asp Val Asn Pro 355 360 365 Asp Glu Ala Val Ala Val
Gly Ala Ala Ile Gln Gly Glu Val Leu Ser 370 375 380 Gly Gly Arg Ser
Asp Val Leu Leu Leu Asp Val Thr Pro Leu Ser Leu 385 390 395 400 Gly
Ile Glu Thr Met Gly Gly Val Met Thr Lys Leu Ile Gln Lys Asn 405 410
415 Thr Thr Ile Pro Thr Lys Ala Ser Gln Val Phe Ser Thr Ala Glu Asp
420 425 430 Asn Gln Ser Ala Val Thr Ile His Val Leu Gln Gly Glu Arg
Glu Arg 435 440 445 Ala Ser Ala Asn Lys Ser Leu Gly Gln Phe Asn Leu
Gly Asp Ile Ala 450 455 460 Pro Ala Pro Arg Gly Met Pro Gln Ile Glu
Val Thr Phe Asp Ile Asp 465 470 475 480 Ala Asn Gly Ile Leu His Val
Ser Ala Lys Asp Lys Gly Thr Gly Lys 485 490 495 Ala Ala Asn Ile Thr
Ile Gln Gly Ser Ser Gly Leu Ser Glu Glu Glu 500 505 510 Ile Glu Arg
Met Val Lys Asp Ala Glu Ala Asn Ala Glu Glu Asp Lys 515 520 525 Lys
Leu Thr Glu Leu Val Ala Ser Arg Asn Gln Ala Glu Ala Leu Ile 530 535
540 His Ser Val Lys Lys Ser Leu Ala Asp Tyr Gly Asp Lys Leu Asp Ala
545 550 555 560 Ala Glu Lys Glu Lys Ile Glu Ala Ala Leu Lys Glu Ala
Glu Glu Ala 565 570 575 Val Lys Gly Asp Asp Lys Ala Ala Ile Asp Ala
Lys Thr Glu Ala Leu 580 585 590 Gly Ala Ala Ser Gln Lys Leu Gly Glu
Met Val Tyr Ala Gln Ala Gln 595 600 605 Ala Glu Ala Gln Ala Gly Glu
Ser Glu Gln Ala Asn Ala Ser Ala Lys 610 615 620 Lys Asp Asp Asp Val
Val Asp Ala Asp Phe Glu Glu Val Lys Asp Asp 625 630 635 640 Lys Lys
16 662 PRT Neisseria meningitidis 16 Met Gly Ser Ser His His His
His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met
Ala Lys Val Ile Gly Ile Asp Leu Gly Thr Thr 20 25 30 Asn Ser Cys
Leu Ala Ile Ser Glu Asn Gly Gln Thr Lys Val Ile Glu 35 40 45 Asn
Ala Glu Gly Ala Arg Thr Thr Pro Ser Val Ile Ala Tyr Leu Asp 50 55
60 Gly Gly Glu Ile Leu Val Gly Ala Pro Ala Lys Arg Gln Ala Val Thr
65 70 75 80 Asn Ala Lys Asn Thr Ile Tyr Ala Ala Lys Arg Leu Ile Gly
His Lys 85 90 95 Phe Glu Asp Lys Glu Val Gln Arg Asp Ile Glu Ser
Met Pro Phe Glu 100 105 110 Ile Ile Lys Ala Asn Asn Gly Asp Ala Trp
Val Lys Ala Gln Gly Lys 115 120 125 Glu Leu Ser Pro Pro Gln Ile Ser
Ala Glu Val Leu Arg Lys Met Lys 130 135 140 Glu Ala Ala Glu Ala Tyr
Leu Gly Glu Lys Val Thr Glu Ala Val Ile 145 150 155 160 Thr Val Pro
Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp 165 170 175 Ala
Gly Arg Ile Ala Gly Leu Asp Val Lys Arg Ile Ile Asn Glu Pro 180 185
190 Thr Ala Ala Ala Leu Ala Phe Gly Met Asp Lys Gly Asp Asn Lys Asp
195 200 205 Arg Lys Val Ala Val Tyr Asp Leu Gly Gly Gly Thr Phe Asp
Ile Ser 210 215 220 Ile Ile Glu Ile Ala Asn Leu Asp Gly Asp Lys Gln
Phe Glu Val Leu 225 230 235 240 Ala Thr Asn Gly Asp Thr Phe Leu Gly
Gly Glu Asp Phe Asp Gln Arg 245 250 255 Leu Ile Asp His Ile Ile Ala
Glu Phe Lys Lys Glu Gln Gly Ile Asp 260 265 270 Leu Lys Gln Asp Val
Met Ala Leu Gln Arg Leu Lys Glu Ala Ala Glu 275 280 285 Lys Ala Lys
Ile Glu Leu Ser Ser Gly Gln Gln Thr Glu Ile Asn Leu 290 295 300 Pro
Tyr Ile Thr Met Asp Ala Thr Gly Pro Lys His Leu Ala Met Lys 305 310
315 320 Ile Thr Arg Ala Lys Phe Glu Ser Leu Val Glu Asp Leu Ile Thr
Arg 325 330 335 Ser Ile Glu Pro Cys Lys Ile Ala Leu Lys Asp Ala Gly
Leu Ser Thr 340 345 350 Gly Asp Ile Asp Asp Val Ile Leu Val Gly Gly
Gln Ser Arg Met Pro 355 360 365 Lys Val Gln Glu Ala Val Lys Ala Phe
Phe Gly Lys Glu Pro Arg Lys 370 375 380 Asp Val Asn Pro Asp Glu Ala
Val Ala Val Gly Ala Ala Ile Gln Gly 385 390 395 400 Glu Val Leu Ser
Gly Gly Arg Ser Asp Val Leu Leu Leu Asp Val Thr 405 410 415 Pro Leu
Ser Leu Gly Ile Glu Thr Met Gly Gly Val Met Thr Lys Leu 420 425 430
Ile Gln Lys Asn Thr Thr Ile Pro Thr Lys Ala Ser Gln Val Phe Ser 435
440 445 Thr Ala Glu Asp Asn Gln Ser Ala Val Thr Ile His Val Leu Gln
Gly 450 455 460 Glu Arg Glu Arg Ala Ser Ala Asn Lys Ser Leu Gly Gln
Phe Asn Leu 465 470 475 480 Gly Asp Ile Ala Pro Ala Pro Arg Gly Met
Pro Gln Ile Glu Val Thr 485 490 495 Phe Asp Ile Asp Ala Asn Gly Ile
Leu His Val Ser Ala Lys Asp Lys 500 505 510 Gly Thr Gly Lys Ala Ala
Asn Ile Thr Ile Gln Gly Ser Ser Gly Leu 515 520 525 Ser Glu Glu Glu
Ile Glu Arg Met Val Lys Asp Ala Glu Ala Asn Ala 530 535 540 Glu Glu
Asp Lys Lys Leu Thr Glu Leu Val Ala Ser Arg Asn Gln Ala 545 550 555
560 Glu Ala Leu Ile His Ser Val Lys Lys Ser Leu Ala Asp Tyr Gly Asp
565 570 575 Lys Leu Asp Ala Ala Glu Lys Glu Lys Ile Glu Ala Ala Leu
Lys Glu 580 585 590 Ala Glu Glu Ala Val Lys Gly Asp Asp Lys Ala Ala
Ile Asp Ala Lys 595 600 605 Thr Glu Ala Leu Gly Ala Ala Ser Gln Lys
Leu Gly Glu Met Val Tyr 610 615 620 Ala Gln Ala Gln Ala Glu Ala Gln
Ala Gly Glu Ser Glu Gln Ala Asn 625 630 635 640 Ala Ser Ala Lys Lys
Asp Asp Asp Val Val Asp Ala Asp Phe Glu Glu 645 650 655 Val Lys Asp
Asp Lys Lys 660 17 37 PRT aspergillus fumigatus 17 Met Gln Arg Ala
Leu Ser Ser Arg Thr Ser Val Leu Ser Ala Ala Ser 1 5 10 15 Lys Arg
Ala Ala Phe Thr Lys Pro Ala Gly Leu Asn Leu Gln Gln Gln 20 25 30
Arg Phe Ala His Lys 35 18 48 PRT aspergillus fumigatus 18 Glu Leu
Lys Phe Gly Val Glu Ala Arg Ala Gln Leu Leu Lys Gly Val 1 5 10 15
Asp Thr Leu Ala Lys Ala Val Thr Ser Thr Leu Gly Pro Lys Gly Arg 20
25 30 Asn Val Leu Ile Glu Ser Pro Tyr Gly Ser Pro Lys Ile Thr Lys
Asp 35 40 45 19 502 PRT aspergillus fumigatus 19 Gly Val Ser Val
Ala Lys Ala Ile Thr Leu Gln Asp Lys Phe Glu Asn 1 5 10 15 Leu Gly
Ala Arg Leu Leu Gln Asp Val Ala Ser Lys Thr Asn Glu Ile 20 25 30
Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Arg Ala Ile Phe 35
40 45 Ser Glu Thr Val Lys Asn Val Ala Ala Gly Cys Asn Pro Met Asp
Leu 50 55 60 Arg Arg Gly Ile Gln Ala Ala Val Asp Ala Val Val Asp
Tyr Leu Gln 65 70 75 80 Lys Asn Lys Arg Asp Ile Thr Thr Gly Glu Glu
Ile Ala Gln Val Ala 85 90 95 Thr Ile Ser Ala Asn Gly Asp Thr His
Ile Gly Lys Leu Ile Ser Thr 100 105 110 Ala Met Glu Arg Val Gly Lys
Glu Gly Val Ile Thr Val Lys Glu Gly 115 120 125 Lys Thr Ile Glu Asp
Glu Leu Glu Val Thr Glu Gly Met Arg Phe Asp 130 135 140 Arg Gly Tyr
Thr Ser Pro Tyr Phe Ile Thr Asp Thr Lys Ser Gln Lys 145 150 155 160
Val Glu Phe Glu Lys Pro Leu Ile Leu Leu Ser Glu Lys Lys Ile Ser 165
170 175 Ala Val Gln Asp Ile Ile Pro Ala Leu Glu Ala Ser Thr Thr Leu
Arg 180 185 190 Arg Pro Leu Val Ile Ile Ala Glu Asp Ile Glu Gly Glu
Ala Leu Ala 195 200 205 Val Cys Ile Leu Asn Lys Leu Arg Gly Gln Leu
Gln Val Ala Ala Val 210 215 220 Lys Ala Pro Gly Phe Gly Asp Asn Arg
Lys Ser Ile Leu Gly Asp Leu 225 230 235 240 Ala Val Leu Thr Asn Gly
Thr Val Phe Thr Asp Glu Leu Asp Ile Lys 245 250 255 Leu Glu Lys Leu
Thr Pro Asp Met Leu Gly Ser Thr Gly Ala Ile Thr 260 265 270 Ile Thr
Lys Glu Asp Thr Ile Ile Leu Asn Gly Glu Gly Ser Lys Asp 275 280 285
Ala Ile Ala Gln Arg Cys Glu Gln Ile Arg Gly Val Met Ala Asp Pro 290
295 300 Ser Thr Ser Glu Tyr Glu Lys Glu Lys Leu Gln Glu Arg Leu Ala
Lys 305 310 315 320 Leu Ser Gly Gly Val Ala Val Ile Lys Val Gly Gly
Ala Ser Glu Val 325 330 335 Glu Val Gly Glu Lys Lys Asp Arg Val Val
Asp Ala Leu Asn Ala Thr 340 345 350 Arg Ala Ala Val Glu Glu Gly Ile
Leu Pro Gly Gly Gly Thr Ala Leu 355 360 365 Leu Lys Ala Ala Ala Asn
Gly Leu Asp Asn Val Lys Pro Glu Asn Phe 370 375 380 Asp Gln Gln Leu
Gly Val Ser Ile Ile Lys Asn Ala Ile Thr Arg Pro 385 390 395 400 Ala
Arg Thr Ile Val Glu Asn Ala Gly Leu Glu Gly Ser Val Ile Val 405 410
415 Gly Lys Leu Thr Asp Glu Phe Ala Lys Asp Phe Asn Arg Gly Phe Asp
420 425 430 Ser Ser Lys Gly Glu Tyr Val Asp Met Ile Ser Ser Gly Ile
Leu Asp 435 440 445 Pro Leu Lys Val Val Arg Thr Ala Leu Leu Asp Ala
Ser Gly Val Ala 450 455 460 Ser Leu Leu Gly Thr Thr Glu Val Ala Ile
Val Glu Ala Pro Glu Glu 465 470 475 480 Lys Gly Pro Ala Ala Pro Gly
Met Gly Gly Met Gly Gly Met Gly Gly 485 490 495 Met Gly Gly Gly Met
Phe 500 20 550 PRT aspergillus fumigatus 20 Met Lys Glu Leu Lys Phe
Gly Val Glu Ala Arg Ala Gln Leu Leu Lys 1 5 10 15 Gly Val Asp Thr
Leu Ala Lys Ala Val Thr Ser Thr Leu Gly Pro Lys 20 25 30 Gly Arg
Asn Val Leu Ile Glu Ser Pro Tyr Gly Ser Pro Lys Ile Thr 35 40 45
Lys Asp Gly Val Ser Val Ala Lys Ala Ile Thr Leu Gln Asp Lys Phe 50
55 60 Glu Asn Leu Gly Ala Arg Leu Leu Gln Asp Val Ala Ser Lys Thr
Asn 65 70 75 80 Glu Ile Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu
Ala Arg Ala 85 90 95 Ile Phe Ser Glu Thr Val Lys Asn Val Ala Ala
Gly Cys Asn Pro Met 100 105 110 Asp Leu Arg Arg Gly Ile Gln Ala Ala
Val Asp Ala Val Val Asp Tyr 115 120 125 Leu Gln Lys Asn Lys Arg Asp
Ile Thr Thr Gly Glu Glu Ile Ala Gln 130 135 140 Val Ala Thr Ile Ser
Ala Asn Gly Asp Thr His Ile Gly Lys Leu Ile 145 150 155 160 Ser Thr
Ala Met Glu Arg Val Gly Lys Glu Gly Val Ile Thr Val Lys 165 170 175
Glu Gly Lys Thr Ile Glu Asp Glu Leu Glu Val Thr Glu Gly Met Arg 180
185 190 Phe Asp Arg Gly Tyr Thr Ser Pro Tyr Phe Ile Thr Asp Thr Lys
Ser 195 200 205 Gln Lys Val Glu Phe Glu Lys Pro Leu Ile Leu Leu Ser
Glu Lys Lys 210 215 220 Ile Ser Ala Val Gln Asp Ile Ile Pro Ala Leu
Glu Ala Ser Thr Thr 225 230 235 240 Leu Arg Arg Pro Leu Val Ile Ile
Ala Glu Asp Ile Glu Gly Glu Ala 245 250 255 Leu Ala Val Cys Ile Leu
Asn Lys Leu Arg Gly Gln Leu Gln Val Ala 260 265 270 Ala Val Lys Ala
Pro Gly Phe Gly Asp Asn Arg Lys Ser Ile Leu Gly 275 280 285 Asp Leu
Ala Val Leu Thr Asn Gly Thr Val Phe Thr Asp Glu Leu Asp 290 295 300
Ile Lys Leu Glu Lys Leu Thr Pro Asp Met Leu Gly Ser Thr Gly Ala 305
310 315 320 Ile Thr Ile Thr Lys Glu Asp Thr Ile Ile Leu Asn Gly Glu
Gly Ser
325 330 335 Lys Asp Ala Ile Ala Gln Arg Cys Glu Gln Ile Arg Gly Val
Met Ala 340 345 350 Asp Pro Ser Thr Ser Glu Tyr Glu Lys Glu Lys Leu
Gln Glu Arg Leu 355 360 365 Ala Lys Leu Ser Gly Gly Val Ala Val Ile
Lys Val Gly Gly Ala Ser 370 375 380 Glu Val Glu Val Gly Glu Lys Lys
Asp Arg Val Val Asp Ala Leu Asn 385 390 395 400 Ala Thr Arg Ala Ala
Val Glu Glu Gly Ile Leu Pro Gly Gly Gly Thr 405 410 415 Ala Leu Leu
Lys Ala Ala Ala Asn Gly Leu Asp Asn Val Lys Pro Glu 420 425 430 Asn
Phe Asp Gln Gln Leu Gly Val Ser Ile Ile Lys Asn Ala Ile Thr 435 440
445 Arg Pro Ala Arg Thr Ile Val Glu Asn Ala Gly Leu Glu Gly Ser Val
450 455 460 Ile Val Gly Lys Leu Thr Asp Glu Phe Ala Lys Asp Phe Asn
Arg Gly 465 470 475 480 Phe Asp Ser Ser Lys Gly Glu Tyr Val Asp Met
Ile Ser Ser Gly Ile 485 490 495 Leu Asp Pro Leu Lys Val Val Arg Thr
Ala Leu Leu Asp Ala Ser Gly 500 505 510 Val Ala Ser Leu Leu Gly Thr
Thr Glu Val Ala Ile Val Glu Ala Pro 515 520 525 Glu Glu Lys Gly Pro
Ala Ala Pro Gly Met Gly Gly Met Gly Gly Met 530 535 540 Gly Gly Met
Gly Gly Met 545 550 21 570 PRT aspergillus fumigatus 21 Met Gly Ser
Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg
Gly Ser His Met Lys Glu Leu Lys Phe Gly Val Glu Ala Arg Ala 20 25
30 Gln Leu Leu Lys Gly Val Asp Thr Leu Ala Lys Ala Val Thr Ser Thr
35 40 45 Leu Gly Pro Lys Gly Arg Asn Val Leu Ile Glu Ser Pro Tyr
Gly Ser 50 55 60 Pro Lys Ile Thr Lys Asp Gly Val Ser Val Ala Lys
Ala Ile Thr Leu 65 70 75 80 Gln Asp Lys Phe Glu Asn Leu Gly Ala Arg
Leu Leu Gln Asp Val Ala 85 90 95 Ser Lys Thr Asn Glu Ile Ala Gly
Asp Gly Thr Thr Thr Ala Thr Val 100 105 110 Leu Ala Arg Ala Ile Phe
Ser Glu Thr Val Lys Asn Val Ala Ala Gly 115 120 125 Cys Asn Pro Met
Asp Leu Arg Arg Gly Ile Gln Ala Ala Val Asp Ala 130 135 140 Val Val
Asp Tyr Leu Gln Lys Asn Lys Arg Asp Ile Thr Thr Gly Glu 145 150 155
160 Glu Ile Ala Gln Val Ala Thr Ile Ser Ala Asn Gly Asp Thr His Ile
165 170 175 Gly Lys Leu Ile Ser Thr Ala Met Glu Arg Val Gly Lys Glu
Gly Val 180 185 190 Ile Thr Val Lys Glu Gly Lys Thr Ile Glu Asp Glu
Leu Glu Val Thr 195 200 205 Glu Gly Met Arg Phe Asp Arg Gly Tyr Thr
Ser Pro Tyr Phe Ile Thr 210 215 220 Asp Thr Lys Ser Gln Lys Val Glu
Phe Glu Lys Pro Leu Ile Leu Leu 225 230 235 240 Ser Glu Lys Lys Ile
Ser Ala Val Gln Asp Ile Ile Pro Ala Leu Glu 245 250 255 Ala Ser Thr
Thr Leu Arg Arg Pro Leu Val Ile Ile Ala Glu Asp Ile 260 265 270 Glu
Gly Glu Ala Leu Ala Val Cys Ile Leu Asn Lys Leu Arg Gly Gln 275 280
285 Leu Gln Val Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Asn Arg Lys
290 295 300 Ser Ile Leu Gly Asp Leu Ala Val Leu Thr Asn Gly Thr Val
Phe Thr 305 310 315 320 Asp Glu Leu Asp Ile Lys Leu Glu Lys Leu Thr
Pro Asp Met Leu Gly 325 330 335 Ser Thr Gly Ala Ile Thr Ile Thr Lys
Glu Asp Thr Ile Ile Leu Asn 340 345 350 Gly Glu Gly Ser Lys Asp Ala
Ile Ala Gln Arg Cys Glu Gln Ile Arg 355 360 365 Gly Val Met Ala Asp
Pro Ser Thr Ser Glu Tyr Glu Lys Glu Lys Leu 370 375 380 Gln Glu Arg
Leu Ala Lys Leu Ser Gly Gly Val Ala Val Ile Lys Val 385 390 395 400
Gly Gly Ala Ser Glu Val Glu Val Gly Glu Lys Lys Asp Arg Val Val 405
410 415 Asp Ala Leu Asn Ala Thr Arg Ala Ala Val Glu Glu Gly Ile Leu
Pro 420 425 430 Gly Gly Gly Thr Ala Leu Leu Lys Ala Ala Ala Asn Gly
Leu Asp Asn 435 440 445 Val Lys Pro Glu Asn Phe Asp Gln Gln Leu Gly
Val Ser Ile Ile Lys 450 455 460 Asn Ala Ile Thr Arg Pro Ala Arg Thr
Ile Val Glu Asn Ala Gly Leu 465 470 475 480 Glu Gly Ser Val Ile Val
Gly Lys Leu Thr Asp Glu Phe Ala Lys Asp 485 490 495 Phe Asn Arg Gly
Phe Asp Ser Ser Lys Gly Glu Tyr Val Asp Met Ile 500 505 510 Ser Ser
Gly Ile Leu Asp Pro Leu Lys Val Val Arg Thr Ala Leu Leu 515 520 525
Asp Ala Ser Gly Val Ala Ser Leu Leu Gly Thr Thr Glu Val Ala Ile 530
535 540 Val Glu Ala Pro Glu Glu Lys Gly Pro Ala Ala Pro Gly Met Gly
Gly 545 550 555 560 Met Gly Gly Met Gly Gly Met Gly Gly Met 565 570
22 568 PRT Candida glabrata 22 Met Leu Arg Ala Val Ala Arg Ser Gln
Val Arg Ser Leu Arg Asn Ala 1 5 10 15 Arg Leu Tyr Ser Ser Phe Lys
Glu Leu Lys Phe Gly Val Glu Gly Arg 20 25 30 Ala Ala Leu Leu Arg
Gly Val Glu Thr Leu Ala Asp Ala Val Ser Ala 35 40 45 Thr Leu Gly
Pro Lys Gly Arg Asn Val Leu Ile Glu Gln Pro Phe Gly 50 55 60 Ala
Pro Lys Ile Thr Lys Asp Gly Val Thr Val Ala Arg Ser Ile Thr 65 70
75 80 Leu Glu Asp Lys Phe Glu Asn Met Gly Ala Lys Leu Leu Gln Glu
Val 85 90 95 Ala Ser Lys Thr Asn Glu Ala Ala Gly Asp Gly Thr Thr
Ser Ala Thr 100 105 110 Val Leu Gly Arg Ala Ile Phe Thr Glu Ser Val
Lys Asn Val Ala Ala 115 120 125 Gly Cys Asn Pro Met Asp Leu Arg Arg
Gly Ser Gln Ala Ala Val Glu 130 135 140 Lys Val Ile Gln Phe Leu Thr
Glu Asn Lys Lys Glu Ile Thr Thr Ser 145 150 155 160 Glu Glu Ile Ala
Gln Val Ala Thr Ile Ser Ala Asn Gly Asp Ala His 165 170 175 Val Gly
Lys Leu Leu Ala Ser Ala Met Glu Lys Val Gly Lys Glu Gly 180 185 190
Val Ile Thr Ile Arg Glu Gly Arg Thr Leu Glu Asp Glu Leu Glu Val 195
200 205 Thr Glu Gly Met Arg Phe Asp Arg Gly Phe Ile Ser Pro Tyr Phe
Ile 210 215 220 Thr Asp Ala Lys Ser Gly Lys Val Glu Phe Glu Lys Pro
Leu Leu Leu 225 230 235 240 Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln
Asp Ile Leu Pro Ala Leu 245 250 255 Glu Leu Ser Asn Gln Ser Arg Arg
Pro Leu Leu Ile Ile Ala Glu Asp 260 265 270 Val Asp Gly Glu Ala Leu
Ala Ala Cys Ile Leu Asn Lys Leu Arg Gly 275 280 285 Gln Val Lys Val
Cys Ala Val Lys Ala Pro Gly Phe Gly Asp Asn Arg 290 295 300 Lys Asn
Ile Leu Gly Asp Val Ala Ile Leu Thr Gly Ser Thr Val Phe 305 310 315
320 Thr Glu Glu Leu Asp Leu Lys Pro Glu Gln Ala Thr Met Glu His Leu
325 330 335 Gly Ser Cys Asp Ser Ile Thr Ile Thr Lys Glu Asp Thr Val
Ile Leu 340 345 350 Asn Gly Asn Gly Ser Lys Asp Ser Ile Gln Glu Arg
Ile Glu Gln Ile 355 360 365 Lys Asn Ser Ile Asp Val Thr Thr Thr Asn
Ser Tyr Glu Lys Glu Lys 370 375 380 Leu Gln Glu Arg Leu Ala Lys Leu
Ser Gly Gly Val Ala Val Ile Arg 385 390 395 400 Val Gly Gly Ala Ser
Glu Val Glu Val Gly Glu Lys Lys Asp Arg Tyr 405 410 415 Asp Asp Ala
Leu Asn Ala Thr Arg Ala Ala Val Glu Glu Gly Ile Leu 420 425 430 Pro
Gly Gly Gly Thr Ala Leu Val Lys Ala Ser Arg Val Leu Asp Glu 435 440
445 Val Lys Thr Glu Asn Phe Asp Gln Lys Leu Gly Val Asp Ile Ile Arg
450 455 460 Lys Ala Ile Thr Arg Pro Ala Lys Gln Ile Ile Glu Asn Ala
Gly Glu 465 470 475 480 Glu Gly Ser Val Ile Val Gly Lys Leu Val Asp
Glu Phe Gly Glu Asp 485 490 495 Phe Ala Lys Gly Tyr Asp Ser Ala Lys
Gly Glu Phe Thr Asp Met Leu 500 505 510 Ala Ala Gly Ile Ile Asp Pro
Phe Lys Val Val Arg Ser Gly Leu Val 515 520 525 Asp Ala Ser Gly Val
Ala Ser Leu Leu Ala Thr Thr Glu Val Ala Ile 530 535 540 Val Asp Ala
Pro Glu Pro Ala Pro Ala Ala Gly Ala Pro Gly Gly Gly 545 550 555 560
Met Pro Gly Met Pro Gly Met Met 565 23 548 PRT Candida glabrata 23
Met Ala Lys Glu Leu Lys Phe Gly Val Glu Gly Arg Ala Ala Leu Leu 1 5
10 15 Arg Gly Val Glu Thr Leu Ala Asp Ala Val Ser Ala Thr Leu Gly
Pro 20 25 30 Lys Gly Arg Asn Val Leu Ile Glu Gln Pro Phe Gly Ala
Pro Lys Ile 35 40 45 Thr Lys Asp Gly Val Thr Val Ala Arg Ser Ile
Thr Leu Glu Asp Lys 50 55 60 Phe Glu Asn Met Gly Ala Lys Leu Leu
Gln Glu Val Ala Ser Lys Thr 65 70 75 80 Asn Glu Ala Ala Gly Asp Gly
Thr Thr Ser Ala Thr Val Leu Gly Arg 85 90 95 Ala Ile Phe Thr Glu
Ser Val Lys Asn Val Ala Ala Gly Cys Asn Pro 100 105 110 Met Asp Leu
Arg Arg Gly Ser Gln Ala Ala Val Glu Lys Val Ile Gln 115 120 125 Phe
Leu Thr Glu Asn Lys Lys Glu Ile Thr Thr Ser Glu Glu Ile Ala 130 135
140 Gln Val Ala Thr Ile Ser Ala Asn Gly Asp Ala His Val Gly Lys Leu
145 150 155 160 Leu Ala Ser Ala Met Glu Lys Val Gly Lys Glu Gly Val
Ile Thr Ile 165 170 175 Arg Glu Gly Arg Thr Leu Glu Asp Glu Leu Glu
Val Thr Glu Gly Met 180 185 190 Arg Phe Asp Arg Gly Phe Ile Ser Pro
Tyr Phe Ile Thr Asp Ala Lys 195 200 205 Ser Gly Lys Val Glu Phe Glu
Lys Pro Leu Leu Leu Leu Ser Glu Lys 210 215 220 Lys Ile Ser Ser Ile
Gln Asp Ile Leu Pro Ala Leu Glu Leu Ser Asn 225 230 235 240 Gln Ser
Arg Arg Pro Leu Leu Ile Ile Ala Glu Asp Val Asp Gly Glu 245 250 255
Ala Leu Ala Ala Cys Ile Leu Asn Lys Leu Arg Gly Gln Val Lys Val 260
265 270 Cys Ala Val Lys Ala Pro Gly Phe Gly Asp Asn Arg Lys Asn Ile
Leu 275 280 285 Gly Asp Val Ala Ile Leu Thr Gly Ser Thr Val Phe Thr
Glu Glu Leu 290 295 300 Asp Leu Lys Pro Glu Gln Ala Thr Met Glu His
Leu Gly Ser Cys Asp 305 310 315 320 Ser Ile Thr Ile Thr Lys Glu Asp
Thr Val Ile Leu Asn Gly Asn Gly 325 330 335 Ser Lys Asp Ser Ile Gln
Glu Arg Ile Glu Gln Ile Lys Asn Ser Ile 340 345 350 Asp Val Thr Thr
Thr Asn Ser Tyr Glu Lys Glu Lys Leu Gln Glu Arg 355 360 365 Leu Ala
Lys Leu Ser Gly Gly Val Ala Val Ile Arg Val Gly Gly Ala 370 375 380
Ser Glu Val Glu Val Gly Glu Lys Lys Asp Arg Tyr Asp Asp Ala Leu 385
390 395 400 Asn Ala Thr Arg Ala Ala Val Glu Glu Gly Ile Leu Pro Gly
Gly Gly 405 410 415 Thr Ala Leu Val Lys Ala Ser Arg Val Leu Asp Glu
Val Lys Thr Glu 420 425 430 Asn Phe Asp Gln Lys Leu Gly Val Asp Ile
Ile Arg Lys Ala Ile Thr 435 440 445 Arg Pro Ala Lys Gln Ile Ile Glu
Asn Ala Gly Glu Glu Gly Ser Val 450 455 460 Ile Val Gly Lys Leu Val
Asp Glu Phe Gly Glu Asp Phe Ala Lys Gly 465 470 475 480 Tyr Asp Ser
Ala Lys Gly Glu Phe Thr Asp Met Leu Ala Ala Gly Ile 485 490 495 Ile
Asp Pro Phe Lys Val Val Arg Ser Gly Leu Val Asp Ala Ser Gly 500 505
510 Val Ala Ser Leu Leu Ala Thr Thr Glu Val Ala Ile Val Asp Ala Pro
515 520 525 Glu Pro Ala Pro Ala Ala Gly Ala Pro Gly Gly Gly Met Pro
Gly Met 530 535 540 Pro Gly Met Met 545 24 568 PRT Candida glabrata
24 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15 Arg Gly Ser His Met Ala Lys Glu Leu Lys Phe Gly Val Glu
Gly Arg 20 25 30 Ala Ala Leu Leu Arg Gly Val Glu Thr Leu Ala Asp
Ala Val Ser Ala 35 40 45 Thr Leu Gly Pro Lys Gly Arg Asn Val Leu
Ile Glu Gln Pro Phe Gly 50 55 60 Ala Pro Lys Ile Thr Lys Asp Gly
Val Thr Val Ala Arg Ser Ile Thr 65 70 75 80 Leu Glu Asp Lys Phe Glu
Asn Met Gly Ala Lys Leu Leu Gln Glu Val 85 90 95 Ala Ser Lys Thr
Asn Glu Ala Ala Gly Asp Gly Thr Thr Ser Ala Thr 100 105 110 Val Leu
Gly Arg Ala Ile Phe Thr Glu Ser Val Lys Asn Val Ala Ala 115 120 125
Gly Cys Asn Pro Met Asp Leu Arg Arg Gly Ser Gln Ala Ala Val Glu 130
135 140 Lys Val Ile Gln Phe Leu Thr Glu Asn Lys Lys Glu Ile Thr Thr
Ser 145 150 155 160 Glu Glu Ile Ala Gln Val Ala Thr Ile Ser Ala Asn
Gly Asp Ala His 165 170 175 Val Gly Lys Leu Leu Ala Ser Ala Met Glu
Lys Val Gly Lys Glu Gly 180 185 190 Val Ile Thr Ile Arg Glu Gly Arg
Thr Leu Glu Asp Glu Leu Glu Val 195 200 205 Thr Glu Gly Met Arg Phe
Asp Arg Gly Phe Ile Ser Pro Tyr Phe Ile 210 215 220 Thr Asp Ala Lys
Ser Gly Lys Val Glu Phe Glu Lys Pro Leu Leu Leu 225 230 235 240 Leu
Ser Glu Lys Lys Ile Ser Ser Ile Gln Asp Ile Leu Pro Ala Leu 245 250
255 Glu Leu Ser Asn Gln Ser Arg Arg Pro Leu Leu Ile Ile Ala Glu Asp
260 265 270 Val Asp Gly Glu Ala Leu Ala Ala Cys Ile Leu Asn Lys Leu
Arg Gly 275 280 285 Gln Val Lys Val Cys Ala Val Lys Ala Pro Gly Phe
Gly Asp Asn Arg 290 295 300 Lys Asn Ile Leu Gly Asp Val Ala Ile Leu
Thr Gly Ser Thr Val Phe 305 310 315 320 Thr Glu Glu Leu Asp Leu Lys
Pro Glu Gln Ala Thr Met Glu His Leu 325 330 335 Gly Ser Cys Asp Ser
Ile Thr Ile Thr Lys Glu Asp Thr Val Ile Leu 340 345 350 Asn Gly Asn
Gly Ser Lys Asp Ser Ile Gln Glu Arg Ile Glu Gln Ile 355 360 365 Lys
Asn Ser Ile Asp Val Thr Thr Thr Asn Ser Tyr Glu Lys Glu Lys 370 375
380 Leu Gln Glu Arg Leu Ala Lys Leu Ser Gly Gly Val Ala Val Ile Arg
385 390 395 400 Val Gly Gly Ala Ser Glu Val Glu Val Gly Glu Lys Lys
Asp Arg Tyr 405 410 415 Asp Asp Ala Leu Asn Ala Thr Arg Ala Ala Val
Glu Glu Gly Ile Leu 420 425 430 Pro Gly Gly Gly Thr Ala Leu Val Lys
Ala Ser Arg Val Leu Asp Glu 435 440 445 Val Lys Thr Glu Asn Phe Asp
Gln Lys Leu Gly Val Asp Ile Ile Arg 450 455 460 Lys Ala Ile Thr Arg
Pro Ala Lys Gln Ile Ile Glu Asn Ala Gly Glu 465 470 475 480 Glu Gly
Ser Val Ile Val Gly Lys Leu Val Asp Glu Phe Gly Glu Asp 485 490 495
Phe Ala Lys Gly Tyr Asp Ser Ala Lys Gly Glu Phe Thr Asp Met Leu 500
505 510 Ala Ala Gly Ile Ile Asp Pro Phe Lys Val Val Arg Ser Gly Leu
Val 515 520 525 Asp Ala Ser
Gly Val Ala Ser Leu Leu Ala Thr Thr Glu Val Ala Ile 530 535 540 Val
Asp Ala Pro Glu Pro Ala Pro Ala Ala Gly Ala Pro Gly Gly Gly 545 550
555 560 Met Pro Gly Met Pro Gly Met Met 565 25 19 DNA Artificial
Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and
to contruct Neisseria meningitidis Hsp70 expression vectors 25
ctgccgtaca tcaccatgg 19 26 19 DNA Artificial Sequence Primer Used
to clone Neisseria meningitidis Hsp70 gene and to contruct
Neisseria meningitidis Hsp70 expression vectors 26 ggcttcttgt
actttcggc 19 27 18 DNA Artificial Sequence Primer Used to clone
Neisseria meningitidis Hsp70 gene and to contruct Neisseria
meningitidis Hsp70 expression vectors 27 tgaccttgtt gaacgtac 18 28
17 DNA Artificial Sequence Primer Used to clone Neisseria
meningitidis Hsp70 gene and to contruct Neisseria meningitidis
Hsp70 expression vectors 28 acttcatcag ggtttac 17 29 6 PRT
Neisseria meningitidis 29 Pro Ala Tyr Phe Asn Asp 1 5 30 18 DNA
Artificial Sequence Primer Used to clone Neisseria meningitidis
Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression
vectors 30 ccngcntayt tyaaygay 18 31 7 PRT Neisseria meningitidis
31 Pro Gln Ile Glu Val Thr Phe 1 5 32 21 DNA Artificial Sequence
Primer Used to clone Neisseria meningitidis Hsp70 gene and to
contruct Neisseria meningitidis Hsp70 expression vectors 32
raangtnacy tcdatytgng g 21 33 16 DNA Artificial Sequence Primer
Used to clone Neisseria meningitidis Hsp70 gene and to contruct
Neisseria meningitidis Hsp70 expression vectors 33 gtaaaacgac
ggccag 16 34 17 DNA Artificial Sequence Primer Used to clone
Neisseria meningitidis Hsp70 gene and to contruct Neisseria
meningitidis Hsp70 expression vectors 34 caggaaacag ctatgac 17 35
27 DNA Artificial Sequence Primer Used to clone Neisseria
meningitidis Hsp70 gene and to contruct Neisseria meningitidis
Hsp70 expression vectors 35 ggtcggctcg ttgatgatgc gtttcac 27 36 27
DNA Artificial Sequence Primer Used to clone Neisseria meningitidis
Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression
vectors 36 gcttctgcca acaaatcttt gggtcag 27 37 24 DNA Artificial
Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and
to contruct Neisseria meningitidis Hsp70 expression vectors 37
gccgctttgg cattcgttat ggac 24 38 24 DNA Artificial Sequence Primer
Used to clone Neisseria meningitidis Hsp70 gene and to contruct
Neisseria meningitidis Hsp70 expression vectors 38 gcgttcgcgt
tcgccttgca gtac 24 39 18 DNA Artificial Sequence Primer Used to
clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria
meningitidis Hsp70 expression vectors 39 ttccgaaaac ggtcaaac 18 40
18 DNA Artificial Sequence Primer Used to clone Neisseria
meningitidis Hsp70 gene and to contruct Neisseria meningitidis
Hsp70 expression vectors 40 atggccaaac aagagttg 18 41 26 DNA
Artificial Sequence Primer Used to clone Neisseria meningitidis
Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression
vectors 41 tacatatggc aaaagtaatc ggtatc 26 42 22 DNA Artificial
Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and
to contruct Neisseria meningitidis Hsp70 expression vectors 42
tttatttttt gtcgtctttt ac 22 43 19 DNA Artificial Sequence Primer
Used to clone Neisseria meningitidis Hsp70 gene and to contruct
Neisseria meningitidis Hsp70 expression vectors 43 gtccaaataa
gcgataacg 19 44 18 DNA Artificial Sequence Primer Used to clone
Neisseria meningitidis Hsp70 gene and to contruct Neisseria
meningitidis Hsp70 expression vectors 44 gccgccaaac gtttgatc 18 45
18 DNA Artificial Sequence Primer Used to clone Neisseria
meningitidis Hsp70 gene and to contruct Neisseria meningitidis
Hsp70 expression vectors 45 accatgggcg gcgtgatg 18 46 18 DNA
Artificial Sequence Primer Used to clone Neisseria meningitidis
Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression
vectors 46 gaagccaatg ccgaggaa 18 47 18 DNA Artificial Sequence
Primer Used to clone Neisseria meningitidis Hsp70 gene and to
contruct Neisseria meningitidis Hsp70 expression vectors 47
tgcgtcgccg ttgttggc 18 48 18 DNA Artificial Sequence Primer Used to
clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria
meningitidis Hsp70 expression vectors 48 ggtatcgccg ttggttgc 18 49
18 DNA Artificial Sequence Primer Used to clone Neisseria
meningitidis Hsp70 gene and to contruct Neisseria meningitidis
Hsp70 expression vectors 49 gagtttgtcg ccgtagtc 18 50 27 DNA
Artificial Sequence Primer Used to clone Aspergillus fumigatus
Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression
plasmids 50 ccatatgaar ganytnaart tyggngt 27 51 20 DNA Artificial
Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and
to contruct Aspergillus fumigatus Hsp60 expression plasmids 51
aanganttna antttggngt 20 52 22 DNA Artificial Sequence Primer Used
to clone Aspergillus fumigatus Hsp60 gene and to contruct
Aspergillus fumigatus Hsp60 expression plasmids 52 cttacatcat
nccnggcatn cc 22 53 19 DNA Artificial Sequence Primer Used to clone
Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus
fumigatus Hsp60 expression plasmids 53 acatcatncc nggcatncc 19 54
25 DNA Artificial Sequence Primer Used to clone Aspergillus
fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60
expression plasmids 54 cttacatncc ncccatnccn cccat 25 55 18 DNA
Artificial Sequence Primer Used to clone Aspergillus fumigatus
Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression
plasmids 55 catnccnccc atnccncc 18 56 20 DNA Artificial Sequence
Primer Used to clone Aspergillus fumigatus Hsp60 gene and to
contruct Aspergillus fumigatus Hsp60 expression plasmids 56
gcnggngayg gnacnacnac 20 57 23 DNA Artificial Sequence Primer Used
to clone Aspergillus fumigatus Hsp60 gene and to contruct
Aspergillus fumigatus Hsp60 expression plasmids 57 ggwccmaagg
ghmgwaatgt ytt 23 58 23 DNA Artificial Sequence Primer Used to
clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus
fumigatus Hsp60 expression plasmids 58 ccnaaratya ctaaggaygg tgt 23
59 20 DNA Artificial Sequence Primer Used to clone Aspergillus
fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60
expression plasmids 59 aarganttna aattyggygt 20 60 20 DNA
Artificial Sequence Primer Used to clone Aspergillus fumigatus
Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression
plasmids 60 tccatnggrt trcanccngc 20 61 20 DNA Artificial Sequence
Primer Used to clone Aspergillus fumigatus Hsp60 gene and to
contruct Aspergillus fumigatus Hsp60 expression plasmids 61
atnacnccyt cyttnccnac 20 62 18 DNA Artificial Sequence Primer Used
to clone Aspergillus fumigatus Hsp60 gene and to contruct
Aspergillus fumigatus Hsp60 expression plasmids 62 catnccytcn
gtnacytc 18 63 23 DNA Artificial Sequence Primer Used to clone
Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus
fumigatus Hsp60 expression plasmids 63 acygartgtg cyattgtyga tgc 23
64 23 DNA Artificial Sequence Primer Used to clone Aspergillus
fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60
expression plasmids 64 acygargttg cyattgtyga tgc 23 65 23 DNA
Artificial Sequence Primer Used to clone Aspergillus fumigatus
Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression
plasmids 65 ttagttgatg cttctggtgt ygc 23 66 23 DNA Artificial
Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and
to contruct Aspergillus fumigatus Hsp60 expression plasmids 66
ttagttgatg ctagyggtgt ygc 23 67 20 DNA Artificial Sequence Primer
Used to clone Aspergillus fumigatus Hsp60 gene and to contruct
Aspergillus fumigatus Hsp60 expression plasmids 67 garaargara
arytncarga 20 68 20 DNA Artificial Sequence Primer Used to clone
Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus
fumigatus Hsp60 expression plasmids 68 gcngcngtng argarggnat 20 69
27 DNA Artificial Sequence Primer Used to clone Aspergillus
fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60
expression plasmids 69 ttacatgccg cccatgccgc ccatacc 27 70 24 DNA
Artificial Sequence Primer Used to clone Aspergillus fumigatus
Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression
plasmids 70 ttacatcata cctggcatac ctgg 24 71 18 DNA Artificial
Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and
to contruct Aspergillus fumigatus Hsp60 expression plasmids 71
ccggtggtga tgtcacgc 18 72 18 DNA Artificial Sequence Primer Used to
clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus
fumigatus Hsp60 expression plasmids 72 ttgatgacgg caacaccg 18 73 18
DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus
Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression
plasmids 73 aactcgtcgg tcagcttg 18 74 18 DNA Artificial Sequence
Primer Used to clone Aspergillus fumigatus Hsp60 gene and to
contruct Aspergillus fumigatus Hsp60 expression plasmids 74
agaacctcgg tgctcgcc 18 75 18 DNA Artificial Sequence Primer Used to
clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus
fumigatus Hsp60 expression plasmids 75 cgccatggag cgtgttgg 18 76 18
DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus
Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression
plasmids 76 tgctgttgag gagggtat 18 77 18 DNA Artificial Sequence
Primer Used to clone Aspergillus fumigatus Hsp60 gene and to
contruct Aspergillus fumigatus Hsp60 expression plasmids 77
atgatgtcct gaacggca 18 78 18 DNA Artificial Sequence Primer Used to
clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus
fumigatus Hsp60 expression plasmids 78 ctgggcgatc ttgccgtc 18 79 21
DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus
Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression
plasmids 79 ggtcgtaacg tccttatcga g 21 80 20 DNA Artificial
Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and
to contruct Aspergillus fumigatus Hsp60 expression plasmids 80
agagtcgaag tcacggcctt 20 81 22 DNA Artificial Sequence Primer Used
to clone Aspergillus fumigatus Hsp60 gene and to contruct
Aspergillus fumigatus Hsp60 expression plasmids 81 cctcaacaat
agcgacctca gt 22 82 18 DNA Artificial Sequence Primer Used to clone
Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus
fumigatus Hsp60 expression plasmids 82 ccccgctgct cctggcat 18 83 19
DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus
Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression
plasmids 83 tcgggcagta gtgttcatc 19 84 33 DNA Artificial Sequence
Primer Used to clone Aspergillus fumigatus Hsp60 gene and to
contruct Aspergillus fumigatus Hsp60 expression plasmids 84
tttctcttct atccttggtg atcttagggg agc 33 85 31 DNA Artificial
Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and
to contruct Aspergillus fumigatus Hsp60 expression plasmids 85
tttctcttca gatggtgtct ctgttgccaa g 31 86 27 DNA Artificial Sequence
Primer Used to clone Aspergillus fumigatus Hsp60 gene and to
contruct Aspergillus fumigatus Hsp60 expression plasmids 86
ttggattcta catcatacct ggcatac 27 87 19 DNA Artificial Sequence
Primer Used to clone Aspergillus fumigatus Hsp60 gene and to
contruct Aspergillus fumigatus Hsp60 expression plasmids 87
taatacgact cactatagg 19 88 19 DNA Artificial Sequence Primer Used
to clone Aspergillus fumigatus Hsp60 gene and to contruct
Aspergillus fumigatus Hsp60 expression plasmids 88 gctagttatt
gctcagcgg 19 89 18 DNA Artificial Sequence Primer Used to clone
Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60
expression plasmids 89 cctatggatt tgagaagg 18 90 18 DNA Artificial
Sequence Primer Used to clone Candida glabrata Hsp60 gene and to
contruct Candida glabrata Hsp60 expression plasmids 90 ctgataatgt
caactccc 18 91 19 DNA Artificial Sequence Primer Used to clone
Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60
expression plasmids 91 gatctcttcc atccaagac 19 92 18 DNA Artificial
Sequence Primer Used to clone Candida glabrata Hsp60 gene and to
contruct Candida glabrata Hsp60 expression plasmids 92 gtccttggag
ccgttacc 18 93 18 DNA Artificial Sequence Primer Used to clone
Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60
expression plasmids 93 ggtaacggct ccaaggac 18 94 19 DNA Artificial
Sequence Primer Used to clone Candida glabrata Hsp60 gene and to
contruct Candida glabrata Hsp60 expression plasmids 94 gtcttggatg
gaagagatc 19 95 18 DNA Artificial Sequence Primer Used to clone
Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60
expression plasmids 95 ccttctcaaa tccatagg 18 96 18 DNA Artificial
Sequence Primer Used to clone Candida glabrata Hsp60 gene and to
contruct Candida glabrata Hsp60 expression plasmids 96 gggagttgac
attatcag 18 97 24 DNA Artificial Sequence Primer Used to clone
Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60
expression plasmids 97 gttgcttcct tgttggctac tacc 24 98 24 DNA
Artificial Sequence Primer Used to clone Candida glabrata Hsp60
gene and to contruct Candida glabrata Hsp60 expression plasmids 98
ccccagcgtg gcagagacag cgtc 24 99 24 DNA Artificial Sequence Primer
Used to clone Candida glabrata Hsp60 gene and to contruct Candida
glabrata Hsp60 expression plasmids 99 gagaacatgg gtgctaagct tctg 24
100 22 DNA Artificial Sequence Primer Used to clone Candida
glabrata Hsp60 gene and to contruct Candida glabrata Hsp60
expression plasmids 100 cagctctgcc ttcgacaccg aa 22 101 23 DNA
Artificial Sequence Primer Used to clone Candida glabrata Hsp60
gene and to contruct Candida glabrata Hsp60 expression plasmids 101
atcaccaagg atggtgtcac cgt 23 102 27 DNA Artificial Sequence Primer
Used to clone Candida glabrata Hsp60 gene and to contruct Candida
glabrata Hsp60 expression plasmids 102 gatatacata tggccaagga
gttgaag 27
* * * * *