U.S. patent application number 15/722949 was filed with the patent office on 2019-04-04 for cpg-oligodeoxynucleotide, immunogenic composition including the same, and method of inducing immune response by the same.
The applicant listed for this patent is National Health Research Institutes. Invention is credited to Tsung-Hsien CHUANG, Chao-Yang LAI, Yi-Ling LIU, Chih-Hao LU, Da-Wei YEH.
Application Number | 20190100756 15/722949 |
Document ID | / |
Family ID | 65895934 |
Filed Date | 2019-04-04 |
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United States Patent
Application |
20190100756 |
Kind Code |
A1 |
CHUANG; Tsung-Hsien ; et
al. |
April 4, 2019 |
CpG-OLIGODEOXYNUCLEOTIDE, IMMUNOGENIC COMPOSITION INCLUDING THE
SAME, AND METHOD OF INDUCING IMMUNE RESPONSE BY THE SAME
Abstract
A CpG-oligodeoxynucleotide (CpG-ODN) for inducing a TLR9
activated immune response, a TLR21 activated immune response or a
combination thereof in a host is provided. The CpG-ODN includes one
or more copies of the sequences of GTCGTT, one or more copies of
the sequences of GTT and one or more copies of the sequences of
TTTT, wherein at least one copy of the sequence of GTCGTT is
encoded between the sequence of GTT and the sequence of TTTT.
Further, an immunogenic composition including the CpG-ODN and a
method of inducing immune response by the same are also
provided.
Inventors: |
CHUANG; Tsung-Hsien; (Miaoli
County, TW) ; YEH; Da-Wei; (Miaoli County, TW)
; LAI; Chao-Yang; (Miaoli County, TW) ; LIU;
Yi-Ling; (Miaoli County, TW) ; LU; Chih-Hao;
(Miaoli County, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Health Research Institutes |
Miaoli County |
|
TW |
|
|
Family ID: |
65895934 |
Appl. No.: |
15/722949 |
Filed: |
October 2, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/117 20130101;
C12N 2310/17 20130101; A61K 39/39 20130101; A61K 31/711 20130101;
A61K 2039/55561 20130101; A61K 31/7088 20130101 |
International
Class: |
C12N 15/117 20060101
C12N015/117; A61K 39/39 20060101 A61K039/39 |
Claims
1. A CpG-oligodeoxynucleotide (CpG-ODN) for inducing a TLR9
activated immune response, a TLR21 activated immune response or a
combination thereof in a host, comprising: one or more copies of
the sequence of GTCGTT; one or more copies of the sequence of GTT;
and one or more copies of the sequence of TTTT; wherein at least
one copy of the sequence of GTCGTT is encoded between the sequence
of GTT and the sequence of TTTT, and the CpG-ODN comprises the
sequence of SEQ ID NO: 1.
2. (canceled)
3. A CpG-oligodeoxynucleotide (CpG-ODN) for inducing a TLR9
activated immune response, a TLR21 activated immune response or a
combination thereof in a host, comprising: one or more copies of
the sequence of GTCGTT; one or more copies of the sequence of GTT;
and one or more copies of the sequence of TTTT; wherein at least
one copy of the sequence of GTCGTT is encoded between the sequence
of GTT and the sequence of TTTT, and the CpG-ODN has a length of 19
nucleotides.
4. A CpG-oligodeoxynucleotide (CpG-ODN) for inducing a TLR9
activated immune response, a TLR21 activated immune response or a
combination thereof in a host, comprising: one or more copies of
the sequence of GTCGTT; one or more copies of the sequence of GTT;
and one or more copies of the sequence of TTTT; wherein at least
one copy of the sequence of GTCGTT is encoded between the sequence
of GTT and the sequence of TTTT, and the CpG-ODN comprises the
sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
5. (canceled)
6. The CpG-ODN as in claim 1, wherein the host is selected from the
group consisting of human, aves, rodent and pisces.
7. An immunogenic composition for inducing a TLR9 activated immune
response, a TLR21 activated immune response or a combination
thereof, comprising: the CpG-ODN as in claim 1; and a vehicle, an
excipient or a combination thereof.
8. The immunogenic composition as in claim 7, further comprising an
antigen selected from the group consisting of: virus, bacterium,
protozoa, and tumor.
9. A method of inducing an immune response of a host for treating
or preventing a disorder, comprising: preparing an immunogenic
composition for inducing a TLR9 activated immune response, a TLR21
activated immune response or a combination thereof, including an
effective dose of a CpG-ODN, wherein the CpG-ODN is as in claim 1;
and administrating the immunogenic composition to the host.
10. The method as in claim 9, wherein the host is selected from the
group consisting of human, aves, rodent and pisces.
11. The method as in claim 9, wherein the effective dose is 0.01
mg/kg body weight to 20 mg/kg body weight.
12. The method as in claim 9, wherein the disorder is selected from
the group consisting of breast cancer, prostate cancer, melanoma,
lymphoma, non-small-cell lung cancer, basal cell carcinoma,
glioblastoma, ovarian cancer, and an infectious disease induced by
hepatitis B virus, B. anthrax, malaria, S. pneumoniae, herpes
simplex virus, influenza virus or a combination thereof.
13. The method as in claim 9, wherein the administrating comprises
administrating orally or by means of injection.
14. An immunogenic composition for inducing a TLR9 activated immune
response, a TLR21 activated immune response or a combination
thereof, comprising: the CpG-ODN as in claim 3; and a vehicle, an
excipient or a combination thereof.
15. An immunogenic composition for inducing a TLR9 activated immune
response, a TLR21 activated immune response or a combination
thereof, comprising: the CpG-ODN as in claim 4; and a vehicle, an
excipient or a combination thereof.
16. A method of inducing an immune response of a host for treating
or preventing a disorder, comprising: preparing an immunogenic
composition for inducing a TLR9 activated immune response, a TLR21
activated immune response or a combination thereof, including an
effective dose of a CpG-ODN, wherein the CpG-ODN is as in claim 3;
and administrating the immunogenic composition to the host.
17. A method of inducing an immune response of a host for treating
or preventing a disorder, comprising: preparing an immunogenic
composition for inducing a TLR9 activated immune response, a TLR21
activated immune response or a combination thereof, including an
effective dose of a CpG-ODN, wherein the CpG-ODN is as in claim 4;
and administrating the immunogenic composition to the host.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0001] The present invention relates to a CpG-oligodeoxynucleotide
(CpG-ODN), an immunogenic composition including the same and
methods of inducing immune response by the same, in particular, to
a CpG-ODN comprising a GTCGTT motif for inducing a TLR9 activated
immune response and/or a TLR21 activated immune response, an
immunogenic composition including the same and a method of
activating such immune responses.
2. Description of the Related Art
[0002] CpG-oligodeoxynucleotides (CpG-ODNs) are known as potent
immune modulators which induce the production of inflammatory
cytokines and a T helper 1 (Th1) polarized immune response,
resulting in the expression of costimulatory molecules in
antigen-presenting cells and increased activation of B cells, T
cells, NK cells and other immunocytes.
[0003] Generally, various CpG-ODNs have species-specific activities
based on the compositions and lengths thereof. For instance,
CpG-1826 (SEQ ID NO: 4) containing two copies of the GACGTT motif
and having a length of 20 nucleotides is more potent in activating
murine cells and less effective in activating human cells than
CpG-2007 (SEQ ID NO: 11) containing three copies of the GTCGTT
motif and having 22 nucleotides. Hence, various kinds of CpG-ODN
have been designed and artificially synthesized for inducing the
immune responses of a specific receptor in a specific species.
[0004] On the other hand, the choices of the target receptor which
is activated by the CpG-ODNs may be also a key point to cost down
and popularize the CpG-ODNs based treatment. A large number of
researchers have found that using Toll-like receptors (TLRs) as a
target induced by the CpG-ODNs in a host may be a better choice
because of its key role in the innate immune system. For instance,
TLR21 in zebrafish (Danio rerio), pufferfish (Takifugu rubripes)
and chicken (Gallus gallus) have shown being able to be activated
by CpG-ODNs (Brownlie R. et al. Molecular immunology. 2009;
46(15):3163-70).
[0005] However, different types of TLRs are expressed in different
species, it may cause unnecessary cost on the developments of
various types of CpG-ODN appropriating to corresponding species.
Hence, developing a single type of CpG-ODN that is able to induce a
plurality of types of TLRs, that is, is able to induce the immune
responses in different kinds of species and has better activation
effects will be a substantial solution of the problem.
SUMMARY OF THE INVENTION
[0006] In the present invention, cells of groupers expressing both
TLR21 and TLR9, mammals expressing TLR9 are used in the test of
activation effects by the CpG-ODNs of the present invention to show
that the CpG-ODN is potent to induce the immune responses in
different species by activating different types of TLRs.
[0007] In the present invention, a CpG-oligodeoxynucleotide
(CpG-ODN) for inducing a TLR9 activated immune response, a TLR21
activated immune response or a combination thereof in a host is
provided. The CpG-ODN comprises one or more copies of the sequence
of GTCGTT, one or more copies of the sequence of GTT and one or
more copies of the sequence of TTTT, wherein at least one copy of
the sequence of GTCGTT is encoded between the sequence of GTT and
the sequence of TTTT.
[0008] Preferably, the CpG-ODN comprises the sequence of SEQ ID NO:
1.
[0009] Preferably, the CpG-ODN has a length of 19 nucleotides.
[0010] Preferably, the CpG-ODN comprises the sequence of SEQ ID NO:
2.
[0011] Preferably, the CpG-ODN comprises the sequence of SEQ ID NO:
3.
[0012] Preferably, the host is selected from a group consisting of
human, aves, rodent and pisces.
[0013] An immunogenic composition for inducing a TLR9 activated
immune response, a TLR21 activated immune response or a combination
thereof, comprising: the CpG-ODNs mentioned above, and a vehicle,
an excipient or a combination thereof.
[0014] Preferably, the immunogenic composition further comprises an
antigen selected from the group consisting of: virus, bacterium,
protozoa, and tumor.
[0015] Additionally, a method of inducing an immune response of a
host for treating or preventing a disorder is provided, comprising:
preparing an immunogenic composition mentioned above which includes
an effective dose of CpG-ODN of the present invention; and
administrating the immunogenic composition to the host.
[0016] Preferably, the host is selected from the group consisting
of human, aves, rodent and pisces.
[0017] Preferably, the disorder is selected from the group
consisting of breast cancer, prostate cancer, melanoma, lymphoma,
non-small-cell lung cancer, basal cell carcinoma, glioblastoma,
ovarian cancer, and an infectious disease induced by hepatitis B
virus, B. anthrax, malaria, S. pneumoniae, herpes simplex virus,
influenza virus or a combination thereof.
[0018] Preferably, the effective dose is 0.01 mg/kg body weight to
20 mg/kg body weight.
[0019] Preferably, the administrating comprises administrating
orally or by means of injection.
[0020] Preferably, the CpG-ODN of present invention is able to
activate both TLR21 and/or TLR9 in a host, that is, is able
inducing immune responses in species of human, aves, rodent,
pisces, and other species expressing TLR21 and/or TLR9.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0022] In the histograms of appended drawings, the statistics
information is as follows: Data represent means.+-.SD (n=3
independent experiments).*P<0.05, **P<0.01 indicates in
comparison with a control group.
[0023] Further, the sequences of the CpG-ODNs labeled in the
drawings are shown in Table 1.
[0024] FIG. 1A shows the computational modeling of the ectodomain
protein structures of orange-spotted grouper (osg, E. coioides) and
giant grouper (gg, E. lanceolatus) TLR21s. FIGS. 1B-1F show the
protein sequence alignment of osgTLR21A, osgTLR21B, and ggTLR21
Toll/interleukin receptor (TIR) domains.
[0025] FIGS. 2A and 2B show the protein identity of the protein
sequences between ggTLR21 and various TLRs referring to the
information obtained from the GenBank database.
[0026] FIG. 3 shows activation of grouper (Epinephelus spp.) TLR21
s induced by different types of CpG-ODNs, wherein part (V) of FIG.
3 shows immunoblot analysis results of the expression of the
grouper TLR21s using .beta.-actin as a loading control.
[0027] FIG. 4 shows histograms illustrating activation of grouper
(Epinephelus spp.) TLR21s induced by different types of CpG-ODNs
and the trimmed derivatives thereof.
[0028] FIG. 5 shows histograms illustrating activation of grouper
(Epinephelus spp.) TLR21s induced by different types of CpG-ODNs
and the trimmed derivatives of the CpG-ODNs in FIG. 3 having higher
activities.
[0029] FIG. 6 shows histograms illustrating activation of grouper
(Epinephelus spp.) TLR21s induced by different types of the trimmed
derivatives of the CpG-ODNs in FIG. 3 having higher activities.
[0030] FIG. 7 shows histograms illustrating activation of grouper
(Epinephelus spp.) TLR21s induced by different types of the trimmed
derivatives of the CpG-ODNs in FIG. 3 having higher activities.
[0031] FIG. 8 shows histograms illustrating activation of grouper
(Epinephelus spp.) TLR21s, human TLR9 and mouse TLR9 induced by
different types of CpG-ODNs and the trimmed derivatives
thereof.
[0032] FIG. 9 shows histograms illustrating induction of different
kinds of cytokine expression in orange-spotted grouper (Epinephelus
coioides) head kidney cells by different types of CpG-ODNs and the
trimmed derivatives thereof.
[0033] FIG. 10 shows histograms illustrating induction of different
kinds of cytokine expression in orange-spotted grouper (Epinephelus
coioides) splenocytes by different types of CpG-ODNs and the
trimmed derivatives thereof.
[0034] FIG. 11 shows histograms illustrating induction of different
kinds of cytokine production in human peripheral blood mononuclear
cells (PBMCs) by different types of CpG-ODNs and the trimmed
derivatives thereof.
[0035] FIG. 12 shows histograms illustrating induction of different
kinds of cytokine production in mouse splenocytes by different
types of CpG-ODNs and the trimmed derivatives thereof.
[0036] FIG. 13 shows histograms illustrating induction of different
kinds of cytokine production in mouse bone marrow-derived
macrophages (BMDMs) by different types of CpG-ODNs and the trimmed
derivatives thereof.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] For the ease of realizing the technical features, contents,
advantages and effects of the present invention by examiners, a
detailed description of the embodiments, the appended drawings and
tables of the present invention will be stated hereinafter. The
drawings are merely used to illustrate and support the content of
the specification and are not intended to be limiting in any way
regarding the implementation of the present invention that must be
explained first.
[0038] Methods and procedures of preparing and obtaining materials
used in the implementation and used for prove the substantial
effects of the present invention will be described below.
[0039] In the embodiments of the present invention, head kidney
cells and splenocytes of groupers were isolated according to the
procedures as follows. Orange-spotted groupers and giant groupers
are anesthetized in water containing 0.2 g/L Tricaine, and the head
kidneys and spleens thereof are aseptically removed. The organs
were then gently minced and passed through a 70-.mu.m strainer with
homogenization buffer (standard Hank's balanced salt solution
(sHBSS) supplemented with 15 mM HEPES, 10% fetal bovine serum
(FBS), 1.times. Antibiotic-Antimycotic, and 50 U/ml heparin).
Subsequently, 1 ml of the homogenized tissue suspension is placed
into 8 ml of distilled water and 1 ml of 10.times.
phosphate-buffered saline (PBS). Following the removal of gross
debris, the cell suspension was centrifuged and washed with
1.times.PBS twice. The cell pellet was then resuspended in
Leibovitz's L-15 cell culture medium (Gibco, Carlsbad, Calif.,
USA).
[0040] Bone marrow-derived macrophages (BMDMs) and splenocytes of
C57BL/6 mice were collected and cultured according to the
procedures as follows. In terms of BMDMs, mouse bone marrow cells
from mouse tibias and femurs were cultured in complete Dulbecco's
Modified Eagle Medium (DMEM) with 30% L929 conditioned medium for 5
days, followed by DMEM medium with 10% FBS. In terms of
splenocytes, they were cultured in RPMI1640 medium with 10% FBS
after obtained. Furthermore, human peripheral blood mononuclear
cells (PBMCs) were cultured in RPMI1640 medium with 10% FBS.
[0041] TLR21 cDNA of giant groupers (ggTLR21 cDNA) were molecular
cloned by procedures as follows. Total RNAs were purified from
grouper splenocytes using TRIzol, and first-strand cDNA libraries
were then prepared using the SuperScript.RTM. III First-Strand
Synthesis System (Invitrogen, Carlsbad, Calif., USA), which is
known in prior arts (Yeh D. W. et al. Proceedings of the National
Academy of Sciences of the United States of America. 2013;
110(51):20711-6). To clone ggTLR21 cDNA, a pair of forward and
reverse primers (5'-GAACAGATTCCTGTACCATGTTCATC-3' (SEQ ID NO: 51)
and 5'-GCTTGTATGAATTGTCACACTGCAC-3' (SEQ ID NO: 52) was designed
based on the sequences of the 5'- and 3'-untranslated regions of
TLR 21 of orange-spotted groupers (osgTLR21) (GenBank: GU198366.2).
A ggTLR21 cDNA containing both of the 5'- and 3'-untranslated
regions and the complete coding region was cloned from the prepared
giant grouper spleen first-strand cDNA library using polymerase
chain reaction (PCR) amplification. Further, the cloned ggTLR21
cDNA sequence is submitted to the GenBank database (accession
number: KM024068).
[0042] Multiple alignments of the amino acid sequences of the
TLR21s were performed using ClustalW2. The structural model of the
TLR21 protein was predicted with SWISS MODEL, using TLR13 as a
template.
[0043] The expression constructs for osgTLR21s and ggTLR21 were
generated through PCR amplification of the corresponding
protein-coding regions from the generated first-strand cDNA
libraries derived from the orange spotted and giant grouper
spleens. The forward and reverse primers based on the 5'- and
3'-end cDNA sequences for the coding region of osgTLR21
(GU198366.2) and ggTLR21 (KM024068) are designed and subcloned the
amplified DNA fragments into a PEF6 vector in frame with a FLAG tag
at their C-terminal ends. The expression vectors for human and
mouse TLR9 were generated following previously described methods
(Liu J. Comparative immunology, microbiology and infectious
diseases. 2012; 35(5):443-51).
[0044] In the embodiments of present invention, TLR21 and TLR9
activation assays are performed according to the procedures as
follows. Human embryonic kidney (HEK) 293 cells were grown in DMEM
supplemented with 10% FBS. Then, the procedures stated in previous
art (Yeh D. W. et al. Proceedings of the National Academy of
Sciences of the United States of America. 2013; 110(51):20711-6)
were performed. Relative luciferase activities were calculated as
the fold change compared with an unstimulated control.
[0045] Reverse transcription-quantitative PCR (RT-qPCR) analysis of
gene expression was performed according to the procedures as
follows. Cells were treated with different CpG-ODNs for 4 h. Then,
the total RNA is purified using TRIzol and the transcription is
performed using, for instance, the SuperScript III First-Strand
Synthesis System (Invitrogen, Carlsbad, Calif., USA). Further,
RT-qPCR was carried out using appropriate detection methods or
kits, for instance, an ABI PRISM 790 reverse 0HT Sequence Detection
System and KAPA SYBR.RTM. fast qPCR kit (KK4605) for gene
expression analysis. The expression of mRNA was normalized to
.beta.-actin. as below. In Table 1, the sequences of comparative
examples are used for explaining the preparation and for comparing
with the CpG-ODN of the examples (SEQ ID NOs: 2-3) of the present
invention so as to show difference on their effects. Further, the
primer sequences used in RT-qPCR are listed in Tables 2 and 3.
TABLE-US-00001 TABLE 1 CpG-ODN SEQUENCE Comparative examples
CpG-1826 TCCATGACGTTCCTGACGTT (SEQ ID NO: 4) CpG-2000
TCCATGACGTTCCTGCAGTTCCTGACGTT (SEQ ID NO: 5) CpG-HC4040
TGACTGTGAACGTTCGAGATGA (SEQ ID NO: 6) CpG-2006
TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 7) CpG-261 TCGTCGTTTTGTCGTT
(SEQ ID NO: 8) CpG-262 GTTTTGTCGTTTTGTCGTT (SEQ ID NO: 9) CpG-263
TCGTTTTGTCGTTTTG (SEQ ID NO: 10) CpG-2007 TCGTCGTTGTCGTTTTGTCGTT
(SEQ ID NO: 11) CpG-271 TCGTCGTTGTCGTTTTGT (SEQ ID NO: 12) CpG-272
GTTGTCGTTTTGTCGTT (SEQ ID NO: 13) CpG-273 TCGTTGTCGTTTTGT (SEQ ID
NO: 14) CpG-2721 GTTGTCGTTGTCGTT (SEQ ID NO: 15) CpG-2723
GTGTCGTTTTGTCGTT (SEQ ID NO: 16) CpG-2724 GTTGTCGTTTTGTC (SEQ ID
NO: 17) CpG-2725 GTTGTCGTTTCC (SEQ ID NO: 18) CpG-2726
GTTGTGCTTTTTTGTCGTT (SEQ ID NO: 19) CpG-2728 GTTGTGCTTTTTTGTGCTT
(SEQ ID NO: 20) CpG-2729 (SEQ ID NO: 21) GTCGTTTTTTGTCGTT CpG-2730
(SEQ ID NO: 22) GTTGTCGTTTTTT Examples of embodiments CpG-2731
GTCGTTTTTT (SEQ ID NO: 1) CpG-2722 GTTGTCGTTTTTTGTCGTT (SEQ ID NO:
2) CpG-2727 GTTGTCGTTTTTTGTGCTT (SEQ ID NO: 3)
TABLE-US-00002 TABLE 2 Grouper primers used in RT-qPCR .beta.-actin
forward 5'-GACATGGTGCGGTTTCTCTT-3' (SEQ ID NO: 23) reverse
5'-GCCTCTGCTGTGCTGATGTA-3' (SEQ ID NO: 24) TNF-.alpha. forward
5'-GGATCTGGCGCTACTCAGAC-3' (SEQ ID NO: 25) reverse
5'-TCCGATAGCTGGTTGGTTTC-3' (SEQ ID NO: 26) IL-1.beta. forward
5'-GACATGGTGCGGTTTCTCTT-3' (SEQ ID NO: 27) reverse
5'-GCCTCTGCTGTGCTGATGTA-3' (SEQ ID NO: 28) IL-6 forward
5'-CCTGAAGGACCTCGACAATC-3' (SEQ ID NO: 29) reverse
5'-TCCTGACAGCCAGACTTCCT-3' (SEQ ID NO: 30) IL-8 forward
5'-GAGCTGCACTGTCGCTGTAT-3' (SEQ ID NO: 31) reverse
5'-TGTTGGCCATGATCCTGTTA-3' (SEQ ID NO: 32) Mx forward
5'-CCATCTGACGCAACTGAGAA-3' (SEQ ID NO: 33) reverse
5'-TCCACCTCGCAAACTCTCTT-3' (SEQ ID NO: 34) IFN1 forward
5'-CTGTGTCCTTCCCGAATCAT-3' (SEQ ID NO: 35) reverse
5'-TGCACAGTACAGGAGCGAAG-3' (SEQ ID NO: 36) IFN forward
5'-GACCACCAAGATGGAGGCTA-3' gamma (SEQ ID NO: 37) reverse
5'-TACCGGTGTTTCCTCAGGTC-3' (SEQ ID NO: 38) CCL4 forward
5'-GTGGTACTGGCCCAAAGAAA-3' (SEQ ID NO: 39) reverse
5'-GGCTGAAGGTCTGACACACA-3' (SEQ ID NO: 40)
TABLE-US-00003 TABLE 3 Mouse primers used in RT-qPCR gapdh forward
5'-ACCCAGAAGACTGTGGATGG-3' (SEQ ID NO: 41) reverse
5'-CACATTGGGGGTAGGAACAC-3' (SEQ ID NO: 42) tnf-.alpha. forward
5'-GGATCTGGCGCTACTCAGAC-3' (SEQ ID NO: 43) reverse
5'-TCCGATAGCTGGTTGGTTTC-3' (SEQ ID NO: 44) il-1.beta. forward
5'-CAGGCAGGCAGTATCACTCA-3' (SEQ ID NO: 45) reverse
5'-AGCTCATATGGGTCCGACAG-3' (SEQ ID NO: 46) il-6 forward
5'-AGTTGCCTTCTTGGGACTGA-3' (SEQ ID NO: 47) reverse
5'-TCCACGATTTCCCAGAGAAC-3' (SEQ ID NO: 48) cxcl1 forward
5'-GCTGGGATTCACCTCAAGAA-3' (SEQ ID NO: 49) reverse
5'-CTTGGGGACACCTTTTAGCA-3' (SEQ ID NO: 50)
[0046] Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and immunoblot analysis are performed according to the
procedures as follows. Cells were lysed with lysis buffer (100 mM
NaCl, 50 mM Tris-Cl (pH 7.5), 0.5 mM ethylenediaminetetraacetic
acid (EDTA), 1% octylphenoxypolyethoxyethanol (NP-40)) containing
complete protease inhibitor cocktail (Roche Life Science,
Indianapolis, Ind., USA). Then, cell lysates were separated by
SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF)
membranes. Subsequently, membranes were incubated with the
indicated antibody and then with horseradish peroxidase
(HRP)-conjugated secondary antibody of which the immunoreactive
bands were visualized using chemiluminescent HRP substrate
(Immobilon Western; Millipore, Temecula, Calif., USA) and the UVP
BioSpectrum Imaging System.
[0047] Human cytokine production results were measured by the
procedures as follows. PBMCs were treated with different CpG-ODNs
for 24 h, and then the culture supernatant is collected for
cytokine measurement. The production of tumor necrosis factor
(TNF)-.alpha., interleukin (IL)-1.beta., IL-12p70, and interferon
(IFN)-.gamma. were measured using ELISA Kits.
[0048] Furthermore, all of the results shown in the present
disclosure are expressed as the mean.+-.SD of three independent
experiments. Statistical analyses were performed using Student's
t-test with a significance level of P<0.05.
[0049] Here, several embodiments will be described with the results
and the appended drawings for of the present invention for ease of
realizing the effects of the CpG-ODNs provided by the present
invention.
[0050] In an embodiment of the present invention, ggTLR21 may be
cloned according to the methods mentioned above. The cDNA from
osgTLR21A and osgTLR21B may be cloned according to the previous
publication (Li Y. W. et al. Cryptocaryon irritans infection. Fish
& shellfish immunology. 2012; 32(3):476-81). The main
difference between their encoded protein sequences is the absence
of four amino acid residues at the C-terminal end of osgTLR21B, as
shown in FIGS. 1B-1F. Based on the expected high identity between
nucleotide sequences of the same gene in giant grouper and
orange-spotted grouper, the inventor designs two primers based on
the 5'- and 3'-untranslated regions of osgTLR21 to clone ggTLR21.
This results in a full-length ggTLR21 cDNA which is homologous to
osgTLR21A but not osgTLR21B.
[0051] Then, the characteristics of the grouper TLR21s used in the
present invention may be analyzed. To compare the grouper TLR21s
with other TLRs, the inventor aligns their protein sequences with
those of different zebrafish TLRs using ClustalW2 and constructed
an evolution tree as shown in FIG. 2A, wherein the proteins
sequences and labels thereof are obtained from the GenBank
database. As results, the protein identity was 98.26% between
ggTLR21 and osgTLR21A, 98.15% between ggTLR21 and osgTLR21B, 54.44%
between zebrafish (zeb) TLR21 and ggTLR21, and 28.30% between
zebTLR22 and ggTLR21A. TLR22 was closest to TLR21 in the evolution
tree, with TLR13 and TLR20f the next closest.
[0052] Further, as shown in FIG. 2B, phylogenetic analysis using
the protein sequences of TLR21s from different fish and avian
species show that the grouper TLR21s were most closely related to
Oplegnathus fasciatus TLR21, followed by Paralichthys olivaceus and
Scophthalmus maximus TLR21s, and were more distantly related to
chicken (Gullas gullas), duck (Anas platyrhynchos), and goose
(Anser cygnoides) TLR21s.
[0053] On the other hand, the sequences and structures of TLR21s
between difference species of groupers may be also analyzed. As
results, ggTLR21 contains 979 amino acid residues, while osgTLR21A
and osgTLR21B contain 979 and 975, respectively. Alignment of these
three protein sequences shows that they contain an extracellular
domain (ectodomain), a transmembrane domain, and a Toll/IL-1 (TIR)
cytosolic domain. They also have 23 copies of leucine-rich repeats
(LRRs) and a C-terminal leucine-rich repeat (LRR-CT) in their
ectodomain. Only 14 of 741 amino acid residues are differed between
the ectodomain of osgTLR21 and ggTLR21. Several three-dimensional
structures of different TLR ectodomains have been resolved in prior
arts, of which TLR13 is phylogenetically closest to TLR21.
Therefore, the inventor predicts the three-dimensional structures
of the osgTLR21 and ggTLR21 ectodomains with SWISS MODEL software
using TLR13 as a template to further examine their difference. This
shows that their ectodomains are relatively similar to
horseshoe-shaped solenoid three-dimensional structures, except for
the extrusion of a small helix structure at the 315-330 amino acid
regions in osgTLR21, as shown in FIG. 1A in which from left to
right is the result of predicted ectodomain structure of osgTLR21
(black), ggTLR21 (white), and superimposition (black: osgTLR21;
white: ggTLR21) of these two ectodomains. (N: N-terminal end, C:
C-terminal end of the ectodomain). Referring to FIGS. 1B-1F, the
three motifs boxes 1-3 that are required for signal transduction of
mammalian TLRs are conserved in the TIR domains of all three
grouper TLR21s. However, osgTLR21B differs from osgTLR21A and
ggTLR21 in that it lacks a four amino acid residue region at the
C-terminal end after box 3 (Asterisk, identical residues; single
dot, conservative substitutions; two dots, highly conservative
substitutions).
[0054] Subsequently, to investigate whether the structural
differences between these grouper TLR21s translates into
differences in their activity in response to different types of
CpG-ODNs stimulation, the inventor co-transfects HEK293 cells with
the expression vector for these TLR21s and the nuclear factor
(NF)-.kappa.B controlled luciferase reporter gene to establish a
cell-based activation assay. Then, the inventor treats these cells
with CpG-ODNs with different hexamer motifs and sequences, as shown
in Table 1, and measures the induced luciferase reporter
activities. As shown in parts (I) to (IV) of FIG. 3, it was found
that osgTLR21A, osgTLR21B, and ggTLR21 experience the same level of
activation by CpG-ODNs containing GTCGTT hexamer motifs (i.e.,
CpG-2006 [SEQ ID NO: 7] and CpG-2007), but do not respond to
CpG-ODNs containing GACGTT and AACGTT hexamer motifs (i.e.,
CpG-1826, CpG-2000 [SEQ ID NO: 5], and CpG-HC4040 [SEQ ID NO: 6]).
The results suggest that all three grouper TLRs are functional and
these minor structural differences do not significantly affect
their response to CpG-ODN stimulation.
[0055] According to the above results, in an attempt to develop
CpG-ODNs for strong activation of grouper TLR21s, the inventor
trims the length of CpG-2006 and CpG-2007 to retain only the left
or right two of the three GTCGTT hexamer motifs to generate CpG-261
(SEQ ID NO: 8) and CpG-271 (SEQ ID NO: 12), and CpG-262 (SEQ ID NO:
9) and CpG-272 (SEQ ID NO: 13), respectively. Further, the inventor
also trims their length to retain only the middle copy (Table 1).
Then, the activities of these CpG-ODNs are determined by an
osgTLR21 and ggTLR21 cell-based activation assay and being compared
with CpG-2006 and CpG-2007. As results shown in parts (I) to (III)
of FIG. 4, it was found that CpG-272 has the best activities toward
the osgTLR21s and ggTLR21 of the hexamer motif to generate CpG-263
(SEQ ID NO: 10) and CpG-273 (SEQ ID NO: 14). Therefore, inventor
further hexamer motifs to generate CpG-2721 (SEQ ID NO: 15),
CpG-2722 (SEQ ID NO: 2), CpG-2723 (SEQ ID NOs: 16), CpG-2724 (SEQ
ID NO: 17) and CpG-2725 (SEQ ID NO: 18) (Table 1). A further
cell-based assay was performed and the results indicate that
CpG-2722 has the best activity toward the osgTLR21s and ggTLR21, as
shown in part (I) to (III) of FIG. 5.
[0056] Since the CpG-hexamer motifs activating the grouper TLR21s
were found in above results, the inventor further investigates
whether the number of CpG-hexamer motifs and structure of the
CpG-2722 influence the activation of grouper TLR21s. In the above
results, CpG-2722 which contains two copies of the GTCGTT hexamer
motif in a length of nineteen nucleotides has the best activity. To
determine whether both copies are required for its activity, the
inventor reverses the CpG-dideoxynucleotides in its 5'- and
3'-CpG-hexamer motif to generate CpG-2726 (SEQ ID NO: 19), CpG-2727
(SEQ ID NO: 3) and CpG-2728 (SEQ ID NO: 20) (Table 1). As the
results shown in part (I) to (III) of FIG. 6 activation assays of
from osgTLR21 and ggTLR21 indicated that the activity of CpG-2727
is as good as that of CpG-2722, whereas CpG-2726 and CpG-2728 were
unable to activate the osgTLR21s and ggTLR21 grouper TLR21s. These
findings suggest that only one copy of the 5'-CpG-hexamer motif is
required for CpG-2722 activity. The CpG-2722 was further trimmed to
generate CpG-2729 (SEQ ID NO: 21) by removing three nucleotides
from 5'-end, CpG-2730 (SEQ ID NO: 22) by removing the
3'-CpG-hexamer motif, and CpG-2731 (SEQ ID NO: 1) by removing both
of the three nucleotides from 5'-end and the 3'-CpG-hexamer motif
(Table 1).
[0057] Furthermore, the activities of these CpG-ODNs were tested
and the results are shown as in parts (I) to (III) of FIG. 7. The
results showed that the three 5'-end nucleotides were required for
the CpG-2722 to strongly active both of the osgTLR21 and ggTLR21,
and although the 3'-CpG-hexamer motif is not required for the
activity of CpG-2722, these nucleotides are required to maintain
the nineteen nucleotide length of CpG-2722 for its activity.
[0058] Additionally, as shown in parts (I) to (IV) of FIG. 8, the
inventor tests the activities of CpG-2722 and CpG-2727 on human (h)
and mouse (m) TLR9s to determine whether they are specific to
grouper TLR21s. Interestingly, in the hTLR9 activation assay,
according to the results, it was found that the activities of
CpG-2722 and CpG-2727 were as good as those of CpG-2006 and
CpG-2007, which have been optimized for the activation of human
cells. Furthermore, in the mTLR9 activation assay, although the
activities of CpG-2722 and CpG-2727 were not as good as that of
CpG-1826, which has been optimized for the activation of mouse
cells, both had better activities toward mTLR9 than CpG-2006 and
CpG-2007.
[0059] Further, the inventor investigates the effects of CpG-2722
and CpG-2727 on the induction of cytokine productions in grouper,
human, and mouse cells to compare their activities on these cells
with that showed on the cell-based activation assays in parts (I)
to (IV) of FIG. 8. Head kidney cells and splenocytes are purified
from orange-spotted groupers, treated with different CpG-ODNs for
determining the induction of the cytokines IL-1.alpha., IL-6, IL-8,
and IFN.gamma.. In consistent with results obtained from the cell
based assays as show in parts (I) to (IV) of FIG. 9 and parts (I)
to (IV) of FIG. 10, it was found that CpG-2722 and CpG-2727 have
greater effects on the activation of cytokine production in
orange-spotted grouper cells than CpG-1826, CpG-2006, and CpG-2007.
In addition, an ELISA analysis is also performed to show that
CpG-2722 and CpG-2727 are as potent as CpG-2006 and CpG-2007 in
inducing cytokine production in human PBMCs, whereas CpG-1826 had
weak activity on these cells, as shown in parts (I) to (IV) of FIG.
11. Furthermore, CpG-2722 and CpG-2727 were found more potent than
the CpG-2006 and 2007 in inducing cytokine expression in mouse
splenocytes and BMDMs as the results shown in parts (I) to (IV) of
FIG. 12 and parts (I) to (IV) of FIG. 13. These findings suggest
that CpG-2722 and CpG-2727, which were developed for grouper
TLR21s, also effectively activate immune responses in human and
mouse cells.
[0060] According to the embodiments and results stated above, more
details of the present invention may be described and explained in
following paragraphs.
[0061] Generally, TLR9 and TLR21 are the cellular receptors for
CpG-ODNs. TLR9 is expressed in both mammalian and fish species, and
has been better studied in terms of the structural requirements for
CpG-ODN to strongly and species-specifically activate this TLR.
CpG-ODNs that have been optimized for humans are currently being
investigated for their application as vaccine adjuvants, as well as
immunotherapies for allergies, infectious diseases, and cancers.
Similarly, CpG-ODNs have also been shown to exert potent
immunostimulatory activities in chicken, duck, and fish, which
contain TLR21 in previous arts, suggesting potential veterinary
uses of CpG-ODNs as immune modulators and vaccine adjuvants.
However, few studies have investigated the structural requirements
for CpG-ODN to activate TLR21, and it remains unclear whether there
is a common structure that will activate both TLR9 and TLR21.
Therefore, in the present invention, the inventor develops CpG-ODNs
for strong activation of TLR21s in grouper, which are considered an
important aquaculture species in Asia due to their rapid growth and
good price, and investigated the interaction between CpG-ODNs,
grouper TLR21s, and human and mouse TLR9s.
[0062] The inventor found that CpG-ODNs that contained the GTCGTT
motif, such as CpG-2006 and CpG-2007, may activates grouper TLR21s,
whereas those that contained the GACGTT and AACGTT motifs, such as
CpG-1826, CpG-2000, and HC4040, does not. Similarly, it has
previously been shown that zebTLR9s broadly recognize CpG-ODNs with
different CpG-hexamer motifs, with those containing the GACGTT or
AACGTT motifs having better activity toward TLR9, and with the
GTCGTT motif having better activity toward zebTLR21 (Yeh D. W. et
al. Proceedings of the National Academy of Sciences of the United
States of America. 2013; 110(51):20711-6). Thus, the grouper TLR21s
have a similar CpG-hexamer recognition profile to zebTLR21.
[0063] In the present invention, two CpG-ODNs that have better
activities toward grouper TLR21s than CpG-2006 and CpG-2007 are
provided. CpG-2722 has a length of 19 deoxynucleotides and contains
two GTCGTT motifs with four spacing nucleotides between them, while
CpG-2727 is a modification of CpG-2722 in which the
CpG-dideoxynucleotides in the GTCGTT motif has been reversed.
Therefore, the good activity of CpG-2722 suggests that one copy of
the GTCGTT motif is sufficient for strong activation of grouper
TLR21s. In a previous study that developed CpG-ODNs that strongly
activate rabbit TLR9, it was found that the length of a CpG-ODN
also affects its activity (Chuang T H, et al. PloS one. 2014;
9(9):e108808). Thus, the fact that CpG-2722 and CpG-2727 contain
the same type of CpG-hexamer as CpG-2006 and CpG-2007, but have
different lengths and numbers of the CpG hexamer suggests that the
key structural elements for designing CpG-ODNs to strongly activate
TLR21 are the same as those for TLR9.
[0064] In the embodiments and results stated above, the activities
of CpG-2722 and CpG-2727 in cells isolated from the head kidneys
and spleens of orange-spotted groupers, which should contain both
TLR9 and TLR21, are also investigated. This shows that although the
activation profile is not entirely the same as for grouper TLR21s,
CpG-2722 and CpG-2727 exhibits a greater effect on cytokine
production in these cells than CpG-2006, CpG-2007, and CpG-1826.
Thus, the CpG-2722 and CpG-2727 may also have good activity toward
grouper TLR9, and it appears that both TLR9 and TLR21 cooperatively
mediate the immunostimulatory activities of CpG-ODNs in groupers,
as previously demonstrated for zebTLR9 and zebTLR21 by CpG-ODNs
with the GTCGTT motif (Yeh D. W. et al. Proceedings of the National
Academy of Sciences of the United States of America. 2013;
110(51):20711-6). Alternatively, this may suggest that TLR21 is the
major receptor for CpG-ODNs in grouper cells.
[0065] On other hand, human and mouse TLR9s have been shown to have
species-specific ligand recognition properties, with human TLR9
being strongly activated by CpG-2006 and CpG-2007, but weakly
activated by CpG-1826, and mouse TLR9 being preferentially
activated by CpG-1826. Here, the inventor found that the activities
of CpG-2722 and CpG-2727 were as strong as those of CpG-2006 and
CpG-2007 in terms of the activation of human TLR9 and cytokine
production in human PBMCs. Furthermore, these two CpG-ODNs also
have better effects than CpG-2006 and CpG-2007 on the activation of
mouse TLR9 and cytokine production in mouse cells. This suggests
that the structures of these two developed CpG-ODNs can better
override the specific-specific ligand recognition properties of
human and mouse TLR9s, and also share a common structure for the
activation of TLR9s and TLR21s from different species. Thus, these
two CpG-ODNs may have potential for use as immunomodulatory or
vaccine adjuvants in a range of species.
[0066] In summary, the present invention provides CpG-ODNs for
inducing a TLR9 activated immune response, a TLR21 activated immune
response or a combination thereof in a host, wherein the CpG-ODNs
comprises one or more copies of the sequence of GTCGTT, one or more
copies of the sequence of GTT and one or more copies of the
sequence of TTTT. Further, at least one copy of the sequence of
GTCGTT is encoded between the sequence of GTT and the sequence of
TTTT.
[0067] Preferably, the sequence of TTTT may be encoded right after
the GTCGTT, that is, may be the sequence of GTCGTTTTTT (SEQ ID NO:
1).
[0068] Preferably, the CpG-ODNs having better activities toward
TLR9 and TLR21 are CpG-2722 (SEQ ID NO: 2) and CpG-2727 (SEQ ID NO:
3) which show better efficiencies in the results described
above.
[0069] The host of which the immune responses is being to be
activated by the CpG-ODNs provided by the present invention may be
species expressing TLR9 and/or TLR21 such as human, aves, rodent
and pisces or any other appropriate species.
[0070] Also, the CpG-ODNs of the present invention may be combined
with a vehicle, and/or an excipient or any other pharmaceutically
acceptable additives as an immunogenic composition to improve the
practicality or biocompatibility thereof.
[0071] Preferably, the immunogenic composition may also be combined
with an antigen to achieve the improvement of the treatment,
specific targeting, or any other additional functions. Preferably,
the antigen may be HBsAg, listeriolysin O, apical membrane antigen
1, MART1, and leishmanial.
[0072] The CpG-ODNs and the immunogenic compositions of the present
invention may also be used in a method of activating an immune
response of a host. The method may comprise steps of preparing an
effective dose of the CpG-ODN or an immunogenic compositions
containing effective dose of the CpG-ODN and administrating the
prepared products into a host.
[0073] In the method of activating an immune response of a host of
the present invention, the effective dose may preferably be 0.01
mg/kg to 20 mg/kg, more preferably 0.1 mg/kg to 20 mg/kg, 0.2 mg/kg
to 10 mg/kg, 0.5 mg/kg to 5 mg/kg.
[0074] Preferably, the host of the present invention may be a
living subject under a demand of a treatment or prevention of a
disorder comprising a cancer, such as breast, prostate, melanoma,
lymphoma, non-small-cell lung cancer, basal cell carcinoma,
glioblastoma, and ovarian cancer and an infectious disease such as
induced by hepatitis B virus, B. anthrax, malaria, S. pneumoniae,
herpes simplex virus, and influenza virus.
[0075] Further, in the method of the present invention, the
administrating may be administrating orally, by means of an
injection or any other general administration methods.
[0076] While the means of specific embodiments in present invention
has been described by reference drawings, numerous modifications
and variations could be made thereto by those skilled in the art
without departing from the scope and spirit of the invention set
forth in the claims. The modifications and variations should in a
range limited by the specification of the present invention.
Sequence CWU 1
1
52110DNAArtificial SequenceAn aspect of the CpG-ODN of the present
invention. 1gtcgtttttt 10219DNAArtificial SequenceAn aspect of the
CpG-ODN of the present invention. 2gttgtcgttt tttgtcgtt
19319DNAArtificial SequenceAn aspect of the CpG-ODN of the present
invention 3gttgtcgttt tttgtgctt 19420DNAArtificial
SequenceCpG-1826, a comparative example of the present invention.
4tccatgacgt tcctgacgtt 20529DNAArtificial SequenceCpG-2000, a
comparative example of the present invention. 5tccatgacgt
tcctgcagtt cctgacgtt 29622DNAArtificial SequenceCpG-HC4040, a
comparative example of the present invention. 6tgactgtgaa
cgttcgagat ga 22724DNAArtificial SequenceCpG-2006, a comparative
example of the present invention. 7tcgtcgtttt gtcgttttgt cgtt
24816DNAArtificial SequenceCpG-261, a comparative example of the
present invention. 8tcgtcgtttt gtcgtt 16919DNAArtificial
SequenceCpG-262, a comparative example of the present invention.
9gttttgtcgt tttgtcgtt 191016DNAArtificial SequenceCpG-263, a
comparative example of the present invention. 10tcgttttgtc gttttg
161122DNAArtificial SequenceCpG-2007, a comparative example of the
present invention. 11tcgtcgttgt cgttttgtcg tt 221218DNAArtificial
SequenceCpG-271, a comparative example of the present invention.
12tcgtcgttgt cgttttgt 181317DNAArtificial SequenceCpG-272, a
comparative example of the present invention. 13gttgtcgttt tgtcgtt
171415DNAArtificial SequenceCpG-273, a comparative example of the
present invention. 14tcgttgtcgt tttgt 151515DNAArtificial
SequenceCpG-2721, a comparative example of the present invention.
15gttgtcgttg tcgtt 151616DNAArtificial SequenceCpG-2723, a
comparative example of the present invention. 16gtgtcgtttt gtcgtt
161714DNAArtificial SequenceCpG-2724, a comparative example of the
present invention. 17gttgtcgttt tgtc 141812DNAArtificial
SequenceCpG-2725, a comparative example of the present invention.
18gttgtcgttt cc 121919DNAArtificial SequenceCpG-2726, a comparative
example of the present invention. 19gttgtgcttt tttgtcgtt
192019DNAArtificial SequenceCpG-2728, a comparative example of the
present invention. 20gttgtgcttt tttgtgctt 192116DNAArtificial
SequenceCpG-2729, a comparative example of the present invention.
21gtcgtttttt gtcgtt 162213DNAArtificial SequenceCpG-2730, a
comparative example of the present invention. 22gttgtcgttt ttt
132320DNAArtificial SequenceA forward primer of grouper ]-actin
used in qRT-PCR. 23gacatggtgc ggtttctctt 202420DNAArtificial
SequenceA reverse primer of grouper ]-actin used in qRT-PCR.
24gcctctgctg tgctgatgta 202520DNAArtificial SequenceA forward
primer of grouper TNF-\ used in qRT-PCR. 25ggatctggcg ctactcagac
202620DNAArtificial SequenceA reverse primer of grouper TNF-\ used
in qRT-PCR. 26tccgatagct ggttggtttc 202720DNAArtificial SequenceA
forward primer of grouper IL-1] used in qRT-PCR. 27gacatggtgc
ggtttctctt 202820DNAArtificial SequenceA reverse primer of grouper
IL-1] used in qRT-PCR. 28gcctctgctg tgctgatgta 202920DNAArtificial
SequenceA forward primer of grouper IL-6 used in qRT-PCR.
29cctgaaggac ctcgacaatc 203020DNAArtificial SequenceA reverse
primer of grouper IL-6 used in qRT-PCR. 30tcctgacagc cagacttcct
203120DNAArtificial SequenceA forward primer of grouper IL-8 used
in qRT-PCR. 31gagctgcact gtcgctgtat 203220DNAArtificial SequenceA
reverse primer of grouper IL-8 used in qRT-PCR. 32tgttggccat
gatcctgtta 203320DNAArtificial SequenceA forward primer of grouper
Mx used in qRT-PCR. 33ccatctgacg caactgagaa 203420DNAArtificial
SequenceA reverse primer of grouper Mx used in qRT-PCR.
34tccacctcgc aaactctctt 203520DNAArtificial SequenceA forward
primer of grouper IFN1 used in qRT-PCR. 35ctgtgtcctt cccgaatcat
203620DNAArtificial SequenceA reverse primer of grouper IFN1 used
in qRT-PCR. 36tgcacagtac aggagcgaag 203720DNAArtificial SequenceA
forward primer of grouper IFN gamma used in qRT-PCR. 37gaccaccaag
atggaggcta 203820DNAArtificial SequenceA reverse primer of grouper
IFN gamma used in qRT-PCR. 38taccggtgtt tcctcaggtc
203920DNAArtificial SequenceA forward primer of grouper CCL4 used
in qRT-PCR. 39gtggtactgg cccaaagaaa 204020DNAArtificial SequenceA
reverse primer of grouper CCL4 used in qRT-PCR. 40ggctgaaggt
ctgacacaca 204120DNAArtificial SequenceA forward primer of mouse
gapdh used in qRT-PCR. 41acccagaaga ctgtggatgg 204220DNAArtificial
SequenceA reverse primer of mouse gapdh used in qRT-PCR.
42cacattgggg gtaggaacac 204320DNAArtificial SequenceA forward
primer of mouse tnf-\ used in qRT-PCR. 43ggatctggcg ctactcagac
204420DNAArtificial SequenceA reverse primer of mouse tnf-\ used in
qRT-PCR. 44tccgatagct ggttggtttc 204520DNAArtificial SequenceA
forward primer of mouse il-1] used in qRT-PCR. 45caggcaggca
gtatcactca 204620DNAArtificial SequenceA reverse primer of mouse
il-1] used in qRT-PCR. 46agctcatatg ggtccgacag 204720DNAArtificial
SequenceA forward primer of mouse il-6 used in qRT-PCR.
47agttgccttc ttgggactga 204820DNAArtificial SequenceA reverse
primer of mouse il-6 used in qRT-PCR. 48tccacgattt cccagagaac
204920DNAArtificial SequenceA forward primer of mouse cxcl1 used in
qRT-PCR. 49gctgggattc acctcaagaa 205020DNAArtificial SequenceA
reverse primer of mouse cxcl1 used in qRT-PCR. 50cttggggaca
ccttttagca 205126DNAArtificial SequenceA forward primer for cloning
ggTLR21 cDNA. 51gaacagattc ctgtaccatg ttcatc 265225DNAArtificial
SequenceA reverse primer for cloning ggTLR21 cDNA. 52gcttgtatga
attgtcacac tgcac 25
* * * * *