U.S. patent application number 13/745086 was filed with the patent office on 2014-05-15 for polypeptide adjuvant composition with thermostability and manufacture thereof.
This patent application is currently assigned to National Tsing Hua University. The applicant listed for this patent is National Tsing Hua University. Invention is credited to Chao-Sheng Cheng, Hsien-Sheng Yin.
Application Number | 20140135479 13/745086 |
Document ID | / |
Family ID | 50682314 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140135479 |
Kind Code |
A1 |
Yin; Hsien-Sheng ; et
al. |
May 15, 2014 |
POLYPEPTIDE ADJUVANT COMPOSITION WITH THERMOSTABILITY AND
MANUFACTURE THEREOF
Abstract
The present invention provides a polypeptide adjuvant
composition with thermostability, which is designed from wild-type
chicken interleikin-1.beta. to construct a new chicken
interleikin-1.beta., named CP-interleikin-1.beta.. The
CP-interleikin-1.beta. having improved heat resistance keeps the
original biological activity, and which helps to develop protein
adjuvant with high efficiency and uses in medical application. The
present invention also provides a method of manufacturing such
polypeptide adjuvant composition.
Inventors: |
Yin; Hsien-Sheng; (Hsinchu,
TW) ; Cheng; Chao-Sheng; (Hsinchu, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Tsing Hua University |
Hsinchu |
|
TW |
|
|
Assignee: |
National Tsing Hua
University
Hsinchu
TW
|
Family ID: |
50682314 |
Appl. No.: |
13/745086 |
Filed: |
January 18, 2013 |
Current U.S.
Class: |
530/351 ;
435/69.52 |
Current CPC
Class: |
C07K 14/545
20130101 |
Class at
Publication: |
530/351 ;
435/69.52 |
International
Class: |
C07K 14/545 20060101
C07K014/545 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 9, 2012 |
TW |
101141785 |
Claims
1. A polypeptide adjuvant composition with thermostability,
consisting essentially of a circular permutation chicken
interleukin-1.beta. encoded by a first fragment having the sequence
of SEQ ID NO: 1 and a second fragment having the sequence of SEQ ID
NO: 2, wherein the first fragment is connected to the second
fragment via a linker sequence, the linker sequence is a
polynucleotide sequence SEQ ID NO: 3 encoding a polypeptide SEQ ID
NO: 4.
2. The polypeptide adjuvant composition with thermostability
according to claim 1, wherein the circular permutation chicken
interleukin-1.beta. has thermotolerance in the range 45.degree. C.
to 60.degree. C.
3. The polypeptide adjuvant composition with thermostability
according to claim 1, wherein the secondary structure of the
circular permutation chicken interleukin-1.beta. has .alpha.-helix
and .beta.-folding, and the .alpha.-helix and .beta.-folding having
a ratio of .alpha.-helix to .beta.-folding is 1.07:1 to 1.35:1.
4. (canceled)
5. (canceled)
6. (canceled)
7. A method of manufacturing a polypeptide adjuvant composition
with thermostability, comprising: obtaining at least one Circular
permutation (CP) site of a chicken interleukin polynucleotide
sequence using Circular permutation method, digesting the chicken
interleukin polynucleotide forming two fragment in CP site, and
amplifying the two fragments by polymerase chain reaction (PCR),
connecting the amplified two fragments via a linker sequence,
transforming into a transformant; and obtaining a circular
permutation interleukin from the transformamt, after centrifuging
and extracting, detecting the bioactivity of circular permutation
interleukin with thermostability.
8. The method for manufacturing a polypeptide adjuvant composition
with thermostability according to claim 7, wherein thermostability
is at 45.degree. C.
9. The method for manufacturing a polypeptide adjuvant composition
with thermostability according to claim 7, wherein the linker
sequence is a polynucleotide SEQ ID NO: 3 encoding a polypeptide
SEQ ID NO: 4.
10. The method for manufacturing a polypeptide adjuvant composition
with thermostability according to claim 7, wherein the transformant
is Escherichia coli.
11. A method of preventing avian disease, comprises administering
an avian vaccine to an avian subject, wherein the avian vaccine
includes a polypeptide adjuvant composition with thermostability
according to claim 1.
12. The method of prevent avian disease of claim 11, wherein the
avian vaccine is a chicken vaccine.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of Taiwan
application Ser. No. 101141785, filed on 9 Nov. 2012. The
disclosure of the Taiwan application is incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a polypeptide adjuvant
composition, and in particular, to a polypeptide adjuvant
composition with thermostability. 2. The Prior Arts
[0004] In March and April 1997, there were outbreaks of H5N1 avian
influenza viruses on several poultry farms in the province of Hong
Kong. These viruses were highly pathogenic in chickens and resulted
in high mortality of infected chickens. During this same period,
the continuing occurrence of infection of humans with influenza
virus led to fears of a deadly influenza pandemic. It resulted in
huge economic impact and financial crisis. The best way to prevent
infection with avian influenza is to avoid sources of exposure
whenever possible. Most bird infections with avian influenza in
poultry farms will be culled to avoid the viruses spreading.
However, in high-density poultry farms, once the high-pathogenic
avian influenza outbreaks, the speed of culling the infected birds
may be less than that of the high-pathogenic avian influenza
spread. Vaccination is the most effective way to prevent infection
and severe outcomes caused by influenza viruses. And it needs to
step up the preventive measures against avian influenza.
[0005] Growth in the veterinary vaccines market is primarily
attributed to augmented public awareness and technological
innovations. Many newly developed vaccines are considered
advantageous over traditional vaccines in terms of production
volumes and immune response. Growing demand for the vaccines and
increased vulnerability to diseases such as avian influenza, is
steering the demand for veterinary vaccines in the emerging
Asia-Pacific regions, Moreover, the altering patterns of diseases
among animals and increased development of resistance to currently
used antimicrobials is compelling manufacturers to invest heavily
in new product developments.
[0006] For prevention of bird infection with avian influenza
viruses, a vaccine with excellent quality may reduce the rate of
the birds infected and the viruses transmitted. And it is very
important to manufacture vaccine adjuvants which can enhance
antigen-specific immune responses. Two main known vaccine adjuvants
in livestock are aluminum-based and oil-based. The adverse effect
of both vaccine adjuvants is chemical-makeup, wherein the
aluminum-based adjuvant can not enhance the specificity of the Th1
cell immune response. Oil-based adjuvant can trigger immune system
response, but it may result in local inflammation and granulomatous
reactions at the site of injection, chronic inflammation, skin
ulceration, local abscess or tissue sloughing, diffuse systemic
granulomas, and it is also unable to enhance the production of
antibodies.
[0007] Recently, the vaccine adjuvant has been improved to low or
free oil-based, such as biological adjuvant and protein adjuvant.
In the past, it is well known that mammalian interleukin-1.beta. is
pro-inflammatory cytokines involved in immune defense against
infection. Interleukin-1.beta. is mainly expressed by monocytes and
macrophages and is synthesized as a precursor. The mature,
bioactive interleukin-1.beta. is produced upon caspase 1-mediated
proteolysis of the precursor. The active interleukin-1.beta. is
then able to interact with type 1 interleukin-1 receptor and induce
the expression of immune-related molecules such as cytokines and
chemokines, thus triggering a cascade of immune responses.
Therefore, interleukin-1.beta. can be used as a vaccine adjuvant
for vaccination to stimulate the secretion of chemotactic cytokine
and the production of antibodies.
[0008] However, most adjuvants are unstable at about 46.degree. C.,
and it needs to store at a lower temperature and to maintain the
bioactivity of antigen and the good physical properties in the
vaccine. However, there are heating steps (the range of 40.degree.
C. to 60.degree. C.) in the manufacturing process of the protein
adjuvant, and it is difficult to breakthrough the barrier of
temperature. In addition, vaccines should be stored at the lower
temperature from the time they are manufactured until the time them
used. Overheating vaccines can cause obvious problem, as the
protein in the vaccine will breakdown (denature) and will not
produce the desired immune response. Therefore, it is necessary to
manufacture a vaccine adjuvant with thermostability.
[0009] Interleukin-1.beta. is mainly as a biological adjuvant, but
its instability is limited the application. The present invention
provides an improved circular permutation chicken
interleukin-1.beta. which is obtained by the combination of
structural biology, biological information and genetic engineering.
Because there are many protein adjuvants unstable at the
temperature above 46.degree. C., and chicken interleukin-1.beta. is
also included. The circular permutation chicken interleukin-1.beta.
of the present invention has been broken through the bather which
has not been overcome by other technology. Furthermore, the present
invention also provides a most suitable condition to manufacture
and purify the circular permutation chicken interleukin-1.beta.,
which is able to facilitate industrial applications.
SUMMARY OF THE INVENTION
[0010] The present invention provides a polypeptide adjuvant
composition with thermostability, consisting essentially of a
circular permutation chicken interleukin-1.beta. (CP IL-1.beta.)
encoded by a first fragment SEQ ID NO: 1 and a second fragment SEQ
ID NO: 2, wherein the first fragment is connected to the second
fragment via a linker sequence, the linker sequence is a
polynucleotide sequence SEQ ID NO: 3 encoding a polypeptide SEQ ID
NO: 4. CP IL-1.beta. of the present invention has thermotolerance
in the range 45.degree. C. to 60.degree. C., and the secondary
structure of CP IL-1.beta. has .alpha.-helix and (3-folding, the
ratio of .alpha.-helix to .beta.-folding is 1.07:1 to 1.35:1.
[0011] One object to this invention is to provide a polypeptide
adjuvant composition with thermostability which can be applied in
an avian vaccine, including but not limited to an avian vaccine,
wherein the chicken vaccine is an avian influenza in chicken.
[0012] Another object to this invention is to provide a method of
manufacturing a polypeptide adjuvant composition with
thermostability, comprising: obtain at least one Circular
permutation (CP) site of a chicken interleukin polynucleotide
sequence using Circular permutation method, digest the chicken
interleukin polynucleotide forming two fragment in CP site, and
amplify the two fragments by polymerase chain reaction (PCR),
connect the amplified two fragments via a linker sequence,
transform into a transformant, wherein the transformant is
Escherichia coli.; and obtain a circular permutation interleukin
from the transformamt, after centrifuging and extracting, detect
the bioactivity of circular permutation interleukin with
thermostability. Wherein the linker sequence is a polynucleotide
SEQ ID NO: 3 encoding a polypeptide SEQ ID NO: 4, and the
thermostability is at 45.degree. C.
[0013] The invention provides methods of increasing thermotolerance
or thermostability of a polypeptide adjuvant composition,
consisting essentially of a circular permutation chicken
interleukin-1.beta. encoded by a first fragment SEQ ID NO: 1 and a
second fragment SEQ ID NO: 2, wherein the first fragment is
connected to the second fragment via a linker sequence, the linker
sequence is a polynucleotide sequence SEQ ID NO: 3 encoding a
polypeptide SEQ ID NO: 4, thereby increasing the thermotolerance or
thermostability of the polypeptide adjuvant composition. In one
aspect, the polypeptide adjuvant composition specific activity can
be thermostable or thermotolerant at a temperature in the range
45.degree. C. to 60.degree. C.
[0014] The present invention provides a CP IL-1.beta. using
Circular permutation method to improve the interleukin-1.beta. of
avian. CP IL-1.beta. is more stable to chemical and heating
treatment compared with WT chicken IL-1.beta., and it facilitate
the subsequent processing. CP IL-1.beta. exhibits bioactivities in
vitro and in vivo, moreover, CP IL-1.beta. retains its bioactivity
after high-temperature treatment, which may allow CP IL-1.beta. to
be used in medical, industrial and vaccine applications, such as an
avian adjuvant. It can solve the secondary structure changed by
heating treatment and reduce the cost of the temperature controlled
transport or store for the vaccine.
[0015] The detailed technology and above preferred embodiments
implemented for the present invention are described in the
following paragraphs accompanying the appended drawings for one
skilled in the art to well appreciate the features of the claimed
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a flow chart of constructing a circular
permutation interleukin (IL)-1.beta. of the present invention using
circular permutation method; chlL1 is the abbreviation of the
fragment of wild type (WT) chicken IL-1.beta..
[0017] FIG. 2 is 12% sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS PAGE) of circular permutation
interleukin-1.beta. (CP IL-1.beta.) and WT chicken IL-1.beta. after
expression and purification.
[0018] FIG. 3 shows the CD spectra of WT chicken IL-1.beta. and CP
IL-1.beta. of the present invention.
[0019] FIG. 4 shows the intrinsic fluorescence of WT chicken
IL-1.beta. and CP IL-1.beta. after 1,8-anilinonaphthalenesulfonate
(ANS)-binding assay.
[0020] FIG. 5A shows CD spectra of WT chicken IL-1.beta. after
heating treatment.
[0021] FIG. 5B shows the CD spectra of CP IL-1.beta. after heating
treatment.
[0022] FIG. 6 depicts the thermal denaturation curves of WT chicken
IL-1.beta. and CP IL-1.beta..
[0023] FIG. 7 depicts the chemical (guanidine-HCl) denaturation
curves of WT chicken IL-1.beta. and CP IL-1.beta..
[0024] FIG. 8 shows expression levels of chemokine K60 of WT
chicken IL-1.beta. and CP IL-1.beta..
[0025] FIG. 9 shows the concentration of plasma cortisol of WT
chicken IL-1.beta., WT human IL-1.beta. and CP IL-1.beta.. The
buffer solution used to dissolve interleukin-1.beta. serves as the
internal control.
[0026] FIG. 10 shows concentration of plasma cortisol of WT chicken
IL-1.beta., WT human IL-1.beta. and CP IL-1.beta. at different
temperature (25.degree. C., 45.degree. C., 50.degree. C.,
55.degree. C. and 65.degree. C.).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0027] The present invention provides a circular permutation
interleukin-1.beta. (CP IL1.beta.) using Circular permutation (CP)
method to improve the polypeptide of wild-type chicken
interleukin-1.beta., consisting essentially of a circular
permutation chicken interleukin-1.beta. encoded by a first fragment
SEQ ID NO: 1 and a second fragment SEQ ID NO: 2, wherein the first
fragment is connected to the second fragment via a linker sequence,
the linker sequence is a polynucleotide sequence SEQ ID NO: 3
encoding a polypeptide SEQ ID NO: 4, and constructing a new
recombinant of chicken interleukin-1.beta., named circular
permutation interleukin-1.beta..
[0028] The present invention of a polypeptide adjuvant composition
with thermostability is more stable at high temperature compared
with wild type (WT) chicken IL-1.beta. and it still retains the
structure of protein at 60.degree. C. Moreover, the polypeptide
adjuvant composition of the present invention can retain its
bioactivity after chemical and heating treatment. It can be used in
therapeutic application such as an avian vaccine adjuvant.
Example 1
Construct the Circular Permutation of Chicken Interleukin
(IL)-1.beta.
[0029] In one embodiment, the flow chart of constructing the
circular permutation interleukin-1.beta. (CP IL-1.beta.) as shown
in FIG. 1. Two fragments of wild type (WT) chicken IL-1.beta. is
amplified by polymerase chain reaction (PCR) as a starting
template. A nucleotide linker sequence SEQ ID NO: 3 is obtained by
twice PCR that encodes a polypeptide sequence SEQ ID NO: 4. The
nucleotide linker sequence links the 5' and 3' terminal end of WT
chicken IL-1.beta. gene. The gene of wild type (WT) chicken
IL-1.beta. with the nucleotide linker sequence is as a template
amplified by a forward primer of WT chicken IL-1.beta. gene SEQ ID
NO: 5 and a reverse primer of the nucleotide linker sequence SEQ ID
NO: 8, a reverse primer of WT chicken IL-1.beta. gene SEQ ID NO: 6
and a forward primer of the nucleotide linker sequence SEQ ID NO:
7, respectively. Both PCR products serve as templates and primers
for extension to obtain an extension production. Final, the
extension productions amplified by PCR using forward primer of WT
chicken IL-1.beta. gene SEQ ID NO: 5 and reverse primer of WT
chicken IL-1.beta. gene SEQ ID NO: 6 to obtain a final product. The
final product having two wild-type chicken polynucleotide fragments
and a linker sequence SEQ ID NO: 3 are cloned into vector pGEMT
(Promega, Wis., USA).
[0030] In addition, two fragments of wild type (WT) chicken
IL-1.beta. as a starting template is using circular permutation
method (Taiwan patent I356,103) to estimate a CP site of chicken
interleukin polynucleotide. A circular permutation
interleukin-1.beta. is generated, which is based on the CP site. To
increase the accuracy of the CP site is to estimate a temperature
factor (B-factor) of protein crystal structure at the same time.
The B-factor is indicated the atoms in the crystal fixed or not,
the higher values of the crystallographic B-factor imply greater
uncertainty in atom positions. After digesting the CP site, two
fragments of wild type (WT) chicken IL-1.beta. is generated, a
first fragment SEQ ID NO: 1 and a second fragment SEQ ID NO: 2.
Primers for the PCR amplifications are designed with nucleoside
sequences appropriate for the new N- and C-terminal end. The
circular permutation genes are individually cloned into vector
pET-28a(+) (Promega, Wis., USA), with a His6 and T7 tag at the
Nterminus The gene sequences are confirmed by DNA sequencing.
[0031] Two fragments of wild type (WT) chicken IL-1.beta. is
amplified by polymerase chain reaction (PCR) as a starting
template, and a nucleotide linker sequence SEQ ID NO: 3 links the
first fragment and the second fragment to construct a circular
permutation interleukin-1.beta. (CP IL-1.beta.). CP IL-1.beta. is
cloned into vector pET-28a(+) (Promega, Wis., USA) to produce and
purify protein.
Example 2
Circular Permutation IL-1.beta. Expression and Purification
2.1 Circular Permutation IL-1.beta. Expression
[0032] Escherichia coli BL21(DE3)-CodonPlus cells (Stratagene,
Amsterdam, The Netherlands) bearing pET-28a(+) containing a gene
for the circular permutation IL-1.beta. mutant are cultured in 500
mL of LB broth, 50 .mu.g/mL ampicillin at 37.degree. C. Isopropyl
.beta.-D-1-thiogalactopyranoside (final concentration, 0.4 mM) is
added to induce protein expression when the OD600 of each culture
reach0.6. After 20 h of incubation, the cells are harvested by
centrifugation (8,000.times.g for 20 mM at 4.degree. C.).
2.2 Circular Permutation IL-1.beta. Purification
[0033] Each cell pellet is suspended in 25 mM Tris-HCl, 500 mM
NaCl, pH 7.4 and lyses by sonication. Each lysate was centrifuged
at 100,000.times.g for 30 min at 4.degree. C., and the recombinant
His-tagged protein is purified using a 100-ml Co.sup.2+ affinity
column (BD Biosciences, Calif.). The column is equilibrated with 25
mM Tris-HCl, 500 mM NaCl, pH 7.9. After loading a protein onto the
column, it is eluted in the same buffer that also contains 75 mM
imidazole. To remove the imidazole and concentrate the protein, a
Centricon Plus 20 centrifugal filter device (Millipore, Billerica,
Mass.) is used. The final products are characterized by 12% (w/v)
SDS-PAGE, and the protein concentrations are determined using
Bio-Rad Bradford Protein Assay kit reagents (Bio-Rad, Calif.) with
bovine serum albumin as the standard.
[0034] As shown in FIG. 2, after the WT chicken IL-1.beta. and the
circular permutation IL-1.beta. of the present invention express
and purify, the molecular weight of WT chicken IL-1.beta. is 21.6
kDa and the molecular weight of the circular permutation IL-1.beta.
is 23.5 kDa by 12% sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS PAGE).
Example 3
Determination of Protein Structure
[0035] The circular permutation IL-1.beta. (CP IL-1.beta.) of the
present invention is obtained by circular permutation, and the
structure of CP IL-1.beta. is further determined by circular
dichroism (CD) spectroscopy and fluorescence
spectrofluorometer.
3.1 Determination of the Circular Permutation IL-1.beta. Secondary
Structure
[0036] To determine the secondary structure and stability of the
circular permutation IL-1.beta., an Aviv 202 circular dichroism
spectropolarimeter (Aviv Biomedical Inc., Lakewood, N.J.) is used
to obtain the CD spectra for CP IL-1.beta. and WT chicken
IL-1.beta.. For secondary structure characterization, far-UV CD
spectra of 10 .mu.M samples in 10 mM potassium phosphate, pH 7.4,
are recorded from 260 to 195 nm at 25.degree. C. using a 1-mm path
length cuvette. Three CD scans for each sample are averaged and are
reported as ellipticity (mdeg).
[0037] In FIG. 3 shows the CD spectra of WT chicken IL-1.beta. at
25.degree. C. A strong signal at 205 nm is indicated more
.beta.-folding structure (.alpha.-helix is 19.6.+-.1.4% and
(3-folding is 26.8.+-.1.4%). In contrast, the CD spectra of CP
IL-1.beta. has been changed, there is a maximum value at 195 nm and
two minimum values at 209 nm and 222 nm It is indicated the
secondary structure of CP IL-1.beta. containing mostly
.alpha.-helix. The secondary structure of CP IL-1.beta. is included
.alpha.-helix 26.3.+-.1.6% and .beta.-folding 21.9.+-.1.2% using
CDNN software (Applied Photophysics, England).
3.2 Determination of the Circular Permutation IL-1.beta. Folded
Structure
[0038] The intrinsic fluorescence of 1 mM solutions of CP
IL-1.beta. and WT chicken IL-1.beta. each in 25 mM Tris-HCl, 100 mM
NaCl, pH 7.4, at 25.degree. C. is measured from 290 nm to 400 nm
using an F-7000 fluorescence spectrofluorometer (Hitachi, Tokyo,
Japan) and a 1-cm path length cuvette (excitation at 280 nm). To
characterize potential interactions between CP IL-1.beta. or WT
IL-1.beta. (10 .mu.M each) and the fluorescent hydrophobic dye,
1,8-anilinonaphthalenesulfonate (20 nM), in 25 mM Tris-HCl, 100 mM
NaCl, pH 7.4, at 25.degree. C., the fluorescence of the dye is
measured from 385 to 600 nm (excitation at 365 nm). Each reported
spectrum is the average of three scans and was plotted using
KaleidaGraph software (Synergy Software, Reading, Pa., USA).
[0039] FIG. 4 shows the intrinsic fluorescence of WT chicken
IL-1.beta. and CP IL-1.beta. after 1,8-anilinonaphthalenesulfonate
(ANS)-binding assay. The intrinsic fluorescence of WT chicken
IL-1.beta. shows maximum excitation wavelength at 326 nm, implying
that aromatic amino acids included tyrosine and tryptophan is
embedded in hydrophobic core. In contrast, the intrinsic
fluorescence of CP IL-1.beta. shows maximum excitation wavelength
at 360 nm, implying that aromatic amino acids is exposed on the
surface area of CP IL-1.beta. and not tightly embedded in
hydrophobic core. Moreover, fluorescence intensity of both is
different, implying that there is different force surrounding
aromatic amino acids. Thus, in the present invention, CP IL-1.beta.
has folding patterns of the three-dimensional structure different
from WT chicken IL-1.beta..
Example 4
Determination of Protein Thermostability and Chemistry Property
[0040] The thermostability of CP IL-1.beta. and WT chicken
IL-1.beta. is determined at temperatures between 4.degree. C. and
96.degree. C. by recording the changes in the ellipticity (.theta.)
at 217 nm of their CD spectra. The temperature is raised in
2.degree. C. increments at a heating rate of 1 .degree. C./min
Prior to recording the ellipticity values, the temperature of the
protein sample is equilibrated for 6 sec.
[0041] FIG. 5A shows the CD spectra of WT chicken IL-1.beta. after
heating process to analyses the thermal stability. In the heating
process, WT chicken IL-1.beta. has been obviously reduced the
ellipticity values. Especially the temperature reaches 65.degree.
C., the secondary structure of WT chicken IL-1.beta. has been
broken, while the curve shows two states transition model. Refer to
the FIG. 6, when the temperature reaching 40.degree. C., the
ellipticity values only change slightly. When the temperature over
40.degree. C., the ellipticity values obviously reduce. Where the
temperature reaching 56.degree. C., the structure of WT chicken
IL-1.beta. expands fully and loses the original structure.
[0042] FIG. 5B shows the CD spectra of CP IL-1.beta. at raised
temperature, and CD spectra almost retain the same structure as
which at 25.degree. C. Refer to the FIG. 6, when the temperature up
to 85.degree. C., the secondary structure of CP IL-1.beta. only has
been broken partly. In the experiment, the secondary structure
reduced slowly at high temperature (over 60.degree. C.).
Accordingly, CP IL-1.beta. of the present invention not only
intensifies secondary structure, but also enhances the thermal
stability due to the secondary structure and folding pattern of
protein changing. CP IL-1.beta. of the present invention can
applied broadly and has the benefits of follow-up process and
industrial high-throughput.
[0043] In addition, the affect of protein folding structure is
determined by chemical material. For the guanidine-HCl unfolding
experiment, protein samples (10 .mu.M) in 10 mM potassium
phosphate, pH 7.4, are mixed with solutions containing various
concentrations of guanidine-HCl (GdnHCl) and their ellipticity
values are measured at 217 nm, 25.degree. C. Three independent
unfolding curves are obtained.
[0044] As sown in FIG. 7, protein unfolding is monitored at
different guanidine-HCl concentrations by following the change in
ellipticity at 217 nm, The fraction unfolded is calculated as
f=(.theta.-.theta..sub.F)/(.theta..sub.U-.theta..sub.F), where
.theta. is the observed ellipticity at 217 nm, at a given
guanidine-HCl concentration, and .theta..sub.U and .theta..sub.F
are ellipticities of the denatured and native states, respectively.
At 1.4 M guanidine-HCl, 50% WT chicken IL-1.beta. gets denatured.
At 1.7 M guanidine-HCl, 50% CP IL-1.beta. of the present invention
gets denatured. In addition, at 0 to 1.0 M guanidine-HCl, CP
IL-1.beta. of the present invention gets denatured more slowly than
WT chicken IL-1.beta.. Thus, CP IL-1.beta. of the present invention
also enhances greater resistance to chemical denaturants.
Example 5
Bioactivity Assay in Vitro
[0045] To characterize the bioactivity of CP IL-1.beta., the mRNA
expression level of the pro-inflammatory chemokine K60 in chicken
fibroblasts is determined
[0046] First, chicken DF-1 fibroblasts (from Dr. Lee, Long-Huw at
National Chung Hsing University Department of veterinary medicine)
are cultured in Dulbecco minimum essential medium (DMEM)
supplemented with 4% (v/v) fetal calf serum. CP IL-1.beta., WT
chicken IL-1.beta. and WT human IL-1.beta. (100 ng/ml each) are
then individually added into a culture and incubated for 2 h. The
K60 mRNA levels were then detected as the measure of bioactivity
using RT-PCR kit reagents (SuperScript III One-Step RT PCR System,
Invitrogen, USA). (.beta.-actin mRNA levels served as the internal
control. The PCR primer sequences of RT PCR are as following: the
forward primer SEQ ID NO: 9 and the reverse primer SEQ ID NO: 10 to
detect the K60 mRNA level, the forward primer SEQ ID NO: 11 and the
reverse primer SEQ ID NO: 12 to detect (3-actin mRNA levels. PCR
products were identified after electrophoresis through 1.5% (w/w)
agarose gels.
[0047] As shown in FIG. 8, the results indicate WT human IL-1.beta.
can not promote chemokine K-60 mRNA expression, even through the
structure of WT human IL-1.beta. is identical to the structure of
WT chicken IL-1.beta.. An obvious agarose-gel band corresponding to
K60 mRNA is present in response to CP IL-1.beta. treatment,
implying that CP IL-1.beta., like WT chicken IL-1.beta., triggers
an immune response in fibroblasts. Accordingly, CP IL-1.beta. of
the present invention not only enhances the thermostability but
also retains the bioactivity as WT chicken IL-1.beta..
Example 6
Bioactivity Assay in Vivo
6.1 Measure Plasma Cortisol Level
[0048] To determine the in vivo activity of CP IL-1.beta., WT
chicken IL-1.beta. and WT human IL-1.beta., each protein is
directly injected into the wing vein of adult chickens, after which
plasma cortisol level is measured.
[0049] CP IL-1.beta., WT chicken IL-1.beta. and WT human IL-1.beta.
are individually injected at a concentration of 10 .mu.g/kg body
mass into the wing vein of adult specific-pathogen-free white
leghorn chickens (Animal Health Research Institute, Taiwan). The
buffer solution (25 mM Tris-HCl, 100 mM NaCl) used to dissolve
interleukin-1.beta. serves as the internal control. After 1 h, the
plasma cortisol level is measured using a Roche E170 Modular
immunoassay analyzer (Roche Diagnostics, Mannheim, Germany).
[0050] The plasma cortisol level is measured according to Roche
E170 Modular immunoassay analyzer protocol. As shown in FIG. 9, the
plasma cortisol levels in chickens are significantly enhanced by
intravenous injections of WT chicken IL-1.beta. and CP IL-1.beta.,
which is not found for human IL-1.beta.. Therefore, CP IL-1.beta.
can induce immune and inflammatory responses in chicken, which is
consistent with the K60 mRNA assay.
6.2 Measure Plasma Cortisol Level at High Temperature
[0051] In addition, to investigate the impact of temperature on the
subsequent ability of WT chicken IL-1.beta. or CP IL-1.beta. to
increase the plasma cortisol level, solutions of proteins are heat
to various temperatures (45.degree. C., 50.degree. C., 55.degree.
C., 65.degree. C.) for 10 min and cool to 25.degree. C. They are
then immediately injected into chickens; the residual activity is
defined as the ratio of the cortisol level after and before the
temperature treatment.
[0052] The animal-use protocol is reviewed and approved by the
Institutional Animal Care and Use Committee (IACUC), National Tsing
Hua University, Taiwan. Three independent experiments for each
protein that used three chickens are performed.
[0053] As shown in FIG. 10, solutions of proteins are heat to
various temperatures (25.degree. C., 45.degree. C., 50.degree. C.,
55.degree. C., 65.degree. C.) and subsequently inject the solution
into chickens to measure the serum cortisol level. The results
reveal that heated-treated CP IL-1.beta. causes strikingly better
immune responses than heat-treated WT chicken IL-1.beta.. When
heat-treated at 50.degree. C., WT chicken IL-1.beta. looses more
than 55% of its activity, and CP IL-1.beta. looses about 1% of its
activity. Even after being exposed to 65.degree. C., WT chicken
IL-1.beta. looses about 76% of its activity, but CP IL-1.beta.
retains more than 80% of its activity. These results demonstrate
that the heat tolerance of CP IL-1.beta. allows it to retain its
bioactivity, thereby enhancing the vaccine potency.
[0054] Therefore, the present invention provides a CP IL-1.beta.
which is more stable to chemical and thermal treatments compared
with WT chicken IL-1.beta.. CP IL-1.beta. exhibits in vitro and in
vivo bioactivities, moreover, CP IL-1.beta. retains its bioactivity
after high-temperature treatment, which may allow CP IL-1.beta. to
be used in therapeutic application such as an avian vaccine.
Sequence CWU 1
1
121381DNAchickenInterleukin-1 1caggggccct cctccagcca gaaagtgagg
ctcaacattg cgctgtaccg gccccgaggc 60ccacggggca gcgctggaac tgggcagatg
ccagtggcac tgggcatcaa gggctacaag 120ctctacatgt cgtgtgtgat
gagcggcacc gagcccacac tgcagctgga ggaagccgac 180gtcatgcggg
acatcgacag cgtcgagctg acccgcttca tcttctaccg cctggacagc
240ccgactgagg gcaccacgcg cttcgagtcg gccgccttcc ccgggtggtt
catctgcacc 300tccctgcagc cccggcagcc cgtgggcatc accaaccaac
ccgaccaggt caacatcgcc 360acctacaagc taagtgggcg c
3812105DNAchickenInterleukin-1 2gcgcccgcct tccgctacac ccgctcacag
tccttcgaca tcttcgacat caaccagaag 60tgcttcgtgc tggagtcacc cacccagctg
gtggccctgc acctc 105378DNAArtificial SequenceLinker Sequence
3ggtaccggtg gttcaggtgg ttcaggtggt tcaggtggtt caggtggctc tggtggctct
60ggtggttcag gtggatcc 78426PRTArtificial SequenceLinker Sequence
4Gly Thr Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
Ser Gly 1 5 10 15Gly Ser Gly Gly Ser Gly Gly Ser 20
25530DNAArtificial SequenceForward primer of WT chicken IL-1
5cgaagcttgc gcccgccttc cgctacaccc 30632DNAArtificial
SequenceReverse primer of WT chicken IL-1 6cgggatccgc gcccacttag
cttgtaggtg gc 32736DNAArtificial SequenceForward primer of the
nucleotide linker sequence 7acctgaacca ccggtaccgc gcccacttag cttgta
36831DNAArtificial SequenceReverse primer of the nucleotide linker
sequence 8ggtggttcag gtggatccgc gcccgccttc c 31918DNAArtificial
SequenceForward primer of chemokine K60 9atgatgggca aggctgta
181018DNAArtificial SequenceReverse primer of chemokine K60
10ttaggatgca gtcttatt 181122DNAArtificial SequenceForward primer of
?-Actin 11atgtttgaga ccttcaacac cc 221223DNAArtificial
SequenceReverse primer of ?-Actin 12atgtcacgca caatttctct ctc
23
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