CN102168088A - T cell immunogen gene TI and applications thereof in foot-and-mouth disease protein subunit vaccine and inactivated vaccine - Google Patents
T cell immunogen gene TI and applications thereof in foot-and-mouth disease protein subunit vaccine and inactivated vaccine Download PDFInfo
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Abstract
The invention discloses a T cell immunogen named TI, comprising multiple T cell epitopes and two general T cell epitopes on foot-and-mouth disease virus VP1, VP4, 3A and 3D proteins. The T cell immunogen has a nucleotide sequence as shown in SEQ ID NO: 1 and simultaneously expresses linking encoding genes of two O and Asia 1 serum type foot-and-mouth disease viruses VP1, wherein the expression product is named OA-VPI. The TI immunogen has good effects of specific liquid induction and cell immune response, is a good immune synergist, and can be used as an effective ingredient in the foot-and-mouth disease protein subunit vaccine and the inactivated vaccine so as to improve the immune effect of the vaccine.
Description
Technical field
The present invention relates to the veterinary drug field, especially a kind of T cellular immunization protogene TI and in fmd protein subunit vaccine and inactivated vaccine, using.
Background technology
Foot and mouth disease (Foot-and-Mouth Disease, FMD) be by foot and mouth disease virus (FMDV) cause a kind of acute, hot, the height contagiousness animal epidemic.Mainly infect artiodactyls such as pig, ox, sheep, classified as the deadly infectious disease that must circulate a notice of by International Office of Epizootics.This disease route of transmission is many, velocity of propagation is fast, once repeatedly is very popular at world wide.
FMDV is Picornaviridae (Picornaviridae); this virus of member of Hostis (Aphthovirus) has seven serotypes; be respectively O, A, C, Asia 1, SAT1, SAT2, SAT3; each serotype can produce a lot of gene hypotypes again, no cross protection reaction between the serotype.The nucleic acid of FMDV is the RNA molecule of sub-thread normal chain, about 8500 bases of genome total length, only contain a big open reading frame, the polyprotein that expression produces cracking step by step produces viral structural protein (VP1, VP2, VP3, VP4) and Nonstructural Protein (Lab, 2A, 2B, 2C, 3A, 3B, 3C, 3D), the structural protein assembling produces the capsid structure of virus.Viral capsid proteins is to induce the main immunogens that produces neutralizing antibody, and structural protein and Nonstructural Protein all contain the T cell antigen epitope of inducing cell immunity.
Sequential analysis by monoclonal antibody and immunologic escape mutant strain in early days studies show that, at least there is independently neutrality epitope of 5 functions at O type FMDV (O1BFS), the G-H ring that the section of forming antigen site mainly comprises VP1 with and the C terminal amino acid, on the VP2 31,70～73,75 and 77 amino acids, 43 and 44 site amino acid on the B-C ring of VP1; 58 amino acids on the VP3, VP1149 amino acids (Crowther et al., 1993) etc. participate in constituting five neutralizing epitopes.These 5 neutralizing epitopes are that all the other are conformational epitope the linear epitope except site 1.Utilize the test of synthetic peptide immunization experiment animal and ox to prove that as if site 1 is the epitope of advantage, but the test-results that has show that the antibody response level at site 2 is higher than site 1 (Samuel, 1997) behind the vaccine immunity; Wherein the immunity at G-H ring site obviously is most important, because different serotypes virus, near the variation amplitude maximum the G-H ring, and also the G-H ring is the position (Grubman and Baxt, 2004) of virus in conjunction with cell receptor.T cell epitope produces higher levels of neutralizing antibody for inducing, as early as possible removing virus has important effect, Cooke and Westover (2008) analysis-by-synthesis the difference of different serotypes virus antigen site areas, the result shows that B-cell and T-cell antigen site areas demonstrate serotype feature clearly, have been found that on the structural protein of FMDV and Nonstructural Protein 3A and 3D and all have the T-cell epitope, some T-cell epitope is more conservative between serotype, and some differs greatly between strain; VP4 wherein, VP1,3A all contains conservative t cell epitope in the 3D albumen, and these epi-positions may be easier in the MHC molecular recognition of host cell expression, thereby be subjected to more concern in the molecular vaccine design.
Summary of the invention
Technical problem to be solved by this invention provides a kind of T cellular immunization protogene TI and uses in fmd protein subunit vaccine and inactivated vaccine.
A kind of T cellular immunization protogene TI, it has the nucleotide sequence of SEQ ID NO:1.
Further, it has the aminoacid sequence of SEQ ID NO:2.
T cellular immunization protogene TI uses in fmd protein subunit vaccine and inactivated vaccine.
The result shows, compares with inactivated vaccine or the independent immune group of OA-VP1, and inactivated vaccine or OA-VP1 immune group mouse all can produce high-caliber specificity neutralizing antibody (P＜0.5) behind the interpolation TI antigen; And the CD4+T cell quantity significantly increases (P＜0.01), IFN-γ generation level significantly raise (P＜0.01).Illustrating that TI antigen has the effect of good inducing specific humoral and cellular immune response, is a kind of good immunostimulant, can be used as a kind of effective ingredient in fmd protein subunit vaccine and the inactivated vaccine, to improve the immune effect of vaccine.
The present invention has designed and synthesized and has comprised two Universal T-cell epitopes and tandem expression gene from a plurality of cell epitopes of foot and mouth disease virus structural protein and Nonstructural Protein; can be former in the hope of this expression product as a kind of T cellular immunization; be used for protein subunit vaccine and inactivated vaccine, the level and the immune duration of protectiveness neutralizing antibody behind the raising vaccine immunity.
In the present invention, the VP1 structural protein that O and 1 two types of Asia have been expressed in the design parallel-series are verified the former immunopotentiation of expressed T cellular immunization as the blank of protein subunit vaccine.Intravital preliminary immunity test result shows mouse, expressed T cellular immunization is former to have good immunopotentiation, be expected to be used for invention fmd protein subunit vaccine or, improve the immune effect of traditional vaccine as the immunopotentiation composition of inactivated vaccine.The present invention also provides a kind of new thinking for the aftosa vaccine invention.
Description of drawings
Fig. 1 is the protein molecular quality standard for the SDS-PAGE detection .M of expressing protein TI, and 1 for not inducing the TI contrast, and 2 is to induce back TI expression product, and 3 is TI abduction delivering culture supernatant, and 4 is the precipitation of TI abduction delivering product, and 5 and 6 is the TI expression product behind the purifying;
Fig. 2 is the protein molecular quality standard for the SDS-PAGE detection .M of expressing protein OA-VP1,1 for not inducing the OA-VP1 contrast, 2 for inducing back OA-VP1 expression product, 3 is OA-VP1 abduction delivering culture supernatant, 4 is the precipitation of OA-VP1 abduction delivering product, and 5 is the OA-VP1 expression product after dialysis concentrates.;
Fig. 3 is that immune serum O type FMDV NAT is measured;
Fig. 4 is that immune serum Asia1 type FMDV NAT is measured;
Fig. 5 organizes mice spleen CD4+T cell for each and CD8+T cell subsets quantity streaming detected result .A～F is respectively 1～6 group, and G is that control group .Q2 district shows the CD4+T cell count, and the Q2-1 district shows the CD8+T cell count.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
1 materials and methods
1.1 reagent
Ni-NTA His
Resins is available from Novagen company; Lipopolysaccharides (LPS) is available from Sigma company; RPMI1640 nutrient solution, foetal calf serum are available from GIBCO company; Penicillin, Streptomycin sulphate mix two anti-available from crystalline substance U.S. company; Mouse lymphocyte parting liquid (EZ-SepTM Mouse1X) is a company available from reaching section; IgG2a-FITC, IgG1-PerCP, CD4-PE, IgG2a-PE, CD3-PerCP, CD8-FITC is available from U.S. company BD; Mo μ se IFN-γ ELISA Kit II detection kit is available from U.S. company BD.
1.2T design that cellular immunization is former and expression
Designed and synthesized T cellular immunization protogene TI: it is by foot and mouth disease virus VP1, VP4,3A, 3D albumen is totally 11 t cell epitope genes, the general t cell epitope gene of Measles virus and from the tandem coding gene of the invasion genomic constitution of Yersinia, link to each other with two glycine between each epi-position, nucleotides sequence is classified SEQ ID NO:1:CCATGGCCAAGTTCGTGGCTGCTTGGACCCTGAAAGCTGCTGCTGGCGGCT CCATCATCAACAACTACTACATGCAGCAGTACCAGAACAGCATGGGG G G CACACAGAACAACGATTGGTTCTCCAAGCTGGCCTCCAGCGCTTTCAGCGGACTGT TTGGAGGAGGAAGGACCCTGCCTACATCCTTCAACTACGG AG GA AGGAGGCAGCACACCGACGTGAGCTTTGGCGGAGCCGCCATCGAGTTCTTTGAAGG GATGGTGCACGATTCCATCAAAGGAGGAGTGGACGTGC TGC CAG TGGAGCACATCCTGTACACAAGGATGATGATCGGCAGATTCTGCGGAGGGGTGGTG GCCAGCGACTACGATCTGGACTTCGAAGCCCTGAAG as
Its protein sequence is SEQ ID NO:2:
AKFVAAWTLKAAA (Universal T-cell epitopes)-GG-VP4
(20-34)-GG-VP4
(62-81)-GG-VP1
(157-165)-GG-VP1
(26-34)-GG-3A
(21-35)-GG-3D
(182-201)-GG-3D
(342-371)-GG-TAKSKKFPSYTATYQF (Invasin), that is:
MAKFVAAWTLKAAAGGSIINNYYMQQYQNSMGGTQNNDWFSKLASSAFSGLFGGGRTLPTSFNYGGRRQHTDVSFGGAAIEFFEGMVHDSIKGGVDVLPVEHILYTRMMIGRFCGGVVASDYDLDFEALKPHFKSLGQTITPADKSGGTAKSKKFPSYTATYQF
Each constitutive protein sequence and function see Table 1.
Sequence and the function of table 1 fusion rotein TI
The synthetic antigenic encoding gene of above-mentioned TI, and be inserted into expression vector pET30a, with this recombinant plasmid transformed e. coli bl21 (DE3) competent cell, male reorganization bacterium overnight incubation in the LB of kalamycin resistance liquid nutrient medium will be identified, be transferred in the LB substratum that contains the 0.05mg/mL kalamycin by 1% then, 37 ℃ are cultured to logarithmic phase (OD600=0.6), add IPTG to final concentration be 1mmol/L, behind 37 ℃ of shaking culture 5h, get expression product 1mL and carry out the SDS-PAGE analysis.
1.3O the tandem expression of type and Asia1 type VP1 gene
The VP1 gene of foot and mouth disease virus O/HN/93 and two representative strains of Asia 1/JS/05 increases respectively, and the series connection of the VP1 encoding gene of two serotypes inserted the pET28a expression vector, with recombinant expression vector transformed into escherichia coli BL21 (DE3) competent cell, carry out abduction delivering, expression product called after OA-VP1 with reference to 1.2 methods.
1.4 proteic purifying and evaluation
The positive reorganization bacterium of correctly expressing target protein is cultivated and the abduction delivering target protein in a large number.Carry out purifying to expressing TI albumen application of nickel ion affinity chromatography post, purification process is according to Ni-NTAHis
The Resins process specifications carries out.The OA-VP1 that expresses forms inclusion body, and the purifying inclusion body is used for follow-up test, and purified product carries out SDS-PAGE and analyzes.
1.5 antigen prepd and animal immune
Adopt the concentration of quantification of protein kit measurement purifying protein, being concentrated into final concentration with PEG then is 400 μ g/mL.Add equal-volume 206 oily adjuvants, aspirate repeatedly with the 10mL syringe and make it complete emulsification and make immunizing antigen.
With totally 70 of the Balc/b mouse in age in 6-8 week, be divided into 7 groups (10/group) at random, the 1st～7 group is respectively two independent immune group of valency seedling, two valency seedlings/1 * TI combined immunization group, two valency seedlings/2 * TI combined immunization group, the independent immune group of OA-VP1, OA-VP1/1 * TI combined immunization group, OA-VP1/2 * TI combined immunization group and negative control group; Inject O-Asia 1 type FMDV inactivated vaccine (production of middle peasant Witter biotechnology company) 100 μ L respectively, O-Asia 1 type inactivated vaccine 100 μ L add TI immunogen 100 μ g, O-Asia 1 type inactivated vaccine 100 μ L add 200 μ g TI immunogens, 100 μ g purifying protein OA-VP1,100 μ g OA-VP1 add 100 μ g TI antigens, 100 μ g OA-VP1 add TI antigen 200 μ g and 100 μ LPBS, see table 2 for details.Carry out the hindlimb muscle injection after inactivated vaccine and the OA-VP1 emulsification, and carry out abdominal injection after the emulsification of TI antigen, mouse is raised in clean isolation environment.
Table 2. antigenic kind of each group's mouse immune and dosage
1.6 serum antibody dynamic monitoring and NAT are measured.
14d, 28d, 35d, 45d extract the eyeball blood sampling to mouse after immunity, and separation of serum adopts the neutralizing antibody level in the microneutralization test mensuration serum.Behind 56 ℃ of deactivation 30min of serum to be checked, get 2 times of serial dilutions of 25 μ L samples in DMEM/High Glucose substratum, each extent of dilution repeats 4 holes and adds in the 96 porocyte culture plates; Add the hoof-and-mouth disease venom (O/HN/93 or Asia1/JS/China/05) that 50 μ L contain 100TCID50 in every hole, act on 1h in 37 ℃ of cell culture incubators; It is the BHK-21 cell suspension of 1 * 105/mL that every then hole adds 50 μ L concentration, places 37 ℃ of 5%CO2 cell culture incubators to cultivate.Behind the 48h at the pathology situation and the record in the every hole of microscopic examination, 72h is with the fixing 30min of 4% Paraformaldehyde 96, wash with water behind the 0.05% methylene blue dye liquor dyeing 30min with 10% formalin solution preparation, the sick cell hole is not blue, pathology takes place and the cell detachment person is not painted.Set up positive during test and negative serum contrast, virus recurrence experimental control, the contrast of serum toxicity and normal cell contrast.Calculate according to the Karber method and can protect 50% cell hole not produce cytopathic highest serum extent of dilution, this extent of dilution is the NAT of this part serum.
1.7 mouse spleen T cell surface CD4, CD8, the detection of CD3
After just exempting from 45 days, mouse is extractd the eyeball bloodletting to cause death, be soaked in 75% the alcohol, in super clean bench, take out mouse spleen, 300 order nylon wires grind, add lymphocyte separation medium 5mL and press the process specifications operation, separate the mouse spleen lymphocyte suspension, and be diluted to 2 * 106/mL with PBS after washing 2 times with the PBS that contains 3%FBS, add rat anti-mouse CD4 monoclonal antibody with the PE mark, the rat anti-mouse CD8 monoclonal antibody of the hamster of PerCP mark anti-mouse CD3 monoclonal antibody and FITC mark, establish the homotype control test pipe of respective markers thing simultaneously, the room temperature lucifuge is hatched 20min, washes twice with PBS, uses 500L PBS resuspended at last, go up machine (FACSAria flow cytometer) in the 4h and detect, with CellQuest software analysis total lymphocyte, the quantity of CD4+ and CD8+ cell subsets.
1.8 the detection of mouse spleen lymphocyte secretion of gamma-IFN
Back 45 days of immunity, get 5 mouse for every group, get the spleen isolated lymphocytes, the splenic lymphocyte of fresh separated is placed 96 orifice plates, 37 ℃ of 5%CO2 constant incubators are cultivated, draw 100mL cell culture fluid supernatant behind the 24h, detect the content of IFN-γ in the mouse spleen lymphocyte culture supernatant according to Mouse IFN-γ ELISA Kit II detection kit (available from U.S. company BD) specification sheets.The drafting of typical curve: the standard substance that Mouse IFN-γ ELISA Kit II detection kit is provided carry out doubling dilution, making its concentration is 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL and 31.3pg/mL, operate back drawing standard curve by process specifications.
1.9 data statistic analysis
Use Microsoft Excel software to carry out the different test of significance of two sample difference in means of different group data.Analyze the immunogenic immune-enhancing effect of TI.
2 results
2.1 the expression of target protein and purifying
Use the expression that SDS-PAGE detects target protein in the reorganization bacterium.Electrophoretogram shows that the reorganization bacterium efficiently expresses the OA-VP1 albumen that has produced about 21kDa TI albumen and 55kDa under the IPTG of 1mmol/L inductive condition, and size conforms to intended purposes albumen size.Application of nickel post affinity chromatography technology purifying is expressed the TI albumen that produces, and OA-VP1 albumen has then been carried out preliminary purification (Fig. 1,2) by the purifying inclusion body.By the thin layer scanning analysis, the TI albumen behind the purifying and the purity of OA-VP1 inclusion body have reached respectively more than 90% and 60%.
2.2 the mensuration of serum NAT
The FMDV neutralizing antibody is the most important evaluation that produces protective immunity, by measure immune serum on cell in and the ability of FMDV, the different vaccine antigens that react that can be indirect are induced the effect that produces protective immunity.The result shows, adopts the VP1 antigen immune mouse of two valency seedlings or two serotypes, and 14d begins just can detect neutralizing antibody after the immunity, and the level of neutralizing antibody constantly raises; The neutralizing antibody level of adding 1 times and 2 times TI antigen immune group is significantly higher than does not add TI antigen group (p＜0.05), add 2 times of TI antigen group neutralizing antibodies and be higher than 1 times of TI antigen group of interpolation, but difference is significantly (p＞0.05) not, can significantly improve inactivated vaccine and protein subunit vaccine institute inductive neutralizing antibody level after interpolation TI immunogen be described.The OA-VP1 immune group can detect the neutralizing antibody (table 3 and table 4) at O type and two serotype FMDV of Asia1 separately, and its level is a little less than separately two valency seedling immune group, but difference is not remarkable, and illustrating with OA-VP1 has immune effect (Fig. 3 and Fig. 4) preferably as the blank of protein subunit vaccine.
Table 3 immune serum FMDV NAT is measured (O type)
Table 4 immune serum FMDV NAT is measured (Asia 1 type)
2.3 mouse spleen T cell surface CD4, CD8, the detection of CD3
To the anti-CD4 of isolating mouse spleen lymphocyte, the fluorescence antibody of CD8 and CD3 dyes, and utilizes the FACSAria flow cytometer to detect the propagation result, obtains data with CellQuest software.In the two-dimentional scatter diagram of preceding scattered light (FSC) and offside scattered light (SSC), mark lymphocyte district P1, at 1 * 104 cell of P1 district inside counting.Utilize the lymphocytic kind of multiparameter flow cytometry.Gather CD3 by different fluorescence channels, the fluorescent signal of CD4 and CD8 positive mark cell is also counted (Fig. 5), accounts for the per-cent of selected P1 cellular regions inner cell sum with CellQuest software analysis CD4+T cell and CD8+T cell.Each organizes cell count and per-cent sees Table 5.As shown in Table 5,6 immune group mouse CD4+T and CD8+T cell number average have tangible rising, have compared very evident difference with negative control group; No matter be inactivated vaccine or OA-VP1 protein subunit vaccine, mouse CD4+T cell proportion has tangible rising after adding the TI antigen immune, the ratio that TI antigen is added OA-VP1 protein subunit vaccine immune group CD8+T cell also raises to some extent, illustrates that adding TI antigen has obvious facilitation for cellular immunization.
Table 5. is respectively tested the mouse spleen CD4 of group
+T cell and CD8
+T cell count and per-cent
2.4 the detection of mouse spleen lymphocyte secretion of gamma-IFN
Calculate the IFN-concentration of respectively organizing in the mouse spleen lymphocyte culture sample according to the typical curve of setting up, the results are shown in Table 6.By table 6 as seen, no matter be deactivation vaccine immune group or OA-VP1 protein subunit vaccine immune group, its γ is significantly higher than negative control group (P＜0.01) in disturbing plain generation level; After adding the TI antigen immune, the splenic lymphocyte of inactivated vaccine and OA-VP1 immune group mouse is after accepting the ConA stimulation, and the amount of secretion IFN-all is significantly higher than and does not add the antigenic immune group of TI, significant difference (P＜0.05); It is higher to add behind the TI antigen of 2 multiple doses the secretion level of IFN-, compares difference extremely significantly (P＜0.01) with deactivation vaccine or the independent immune group of OA-VP1.By measuring the generation level of different immune group IFN-, further illustrate and add the level that TI antigen can improve cellular immunization significantly.
Table 6. is respectively tested IFN-γ content in group's mouse spleen lymphocyte culture supernatant
3 discuss
Vaccine inoculation is the reliable and effective means of specificity prevention FMD, and vaccine is a prerequisite of successfully preventing, control and even finally eliminate FMD safely and effectively.Conventional vaccines such as FMD attenuated vaccine and inactivated vaccine all have good immunogenicity, in the process of prevention and control FMD, bringing into play important effect, but, impel people to seek a kind of vaccine of FMD more safely and effectively because virus virulence returns by force, inactivation of virus is not thorough, viral escape goes out unsafe factors such as production of vaccine factory.
Utilize various expression systems to express the main immunogens albumen of FMDV, the development genetic engineering subunit vaccine is the focus of studying at present.Because subunit vaccine only contains the part of pathogenic agent, can not cause the animal morbidity, aspect biological safety, be better than traditional vaccine.Studies show that, structural protein VP1 the 141st～160 amino acid and 200～213 amino acid sections are two advantage neutralizing epitopes of FMDV, the research of a lot of FMDV epiposition vaccines has all comprised above two main epitopes, and has obtained immune effect [7-9] preferably.Wherein, the formed G-H ring of VP1141-160 amino acids is exposed to the surface of virus particle; it is of paramount importance antigenic determinant; this section is widely different between the different pedigree strains of different serotypes and same serotype, is to cause different vaccine strains to lack the principal element of cross immunity protectiveness.Aspect the research of foot-and-mouth disease gene engineering subunit vaccine, how efficiently expressing the virus antigen albumen that spectrotype is wide, immunogenicity is strong is the crucial difficult problem that needs solve.
The fusion VP1 albumen of O type and the two serotypes of Asia1 type has been expressed in design in this research, and design to have expressed in a kind of FMDV of comprising structural protein and the Nonstructural Protein T cellular immunization of a plurality of t cell epitopes and two Universal T-cell epitopes former, expectation is developed a kind of novel fmd protein subunit vaccine with the VP1 albumen and the former combination of this T cellular immunization of two serotypes.By immunity test mouse, preliminary proof, the VP1 albumen of two serotypes can stimulate the neutralizing antibody of mouse generation at two serotypes, and the T cellular immunization of add expressing former after further the immune stimulatory mouse produce higher levels of humoral and cellular immune response, illustrate that cellular immunization and humoral immunization have synergetic property, also explanation employing T cellular immunization is former has good immune effect with the immunity of major antigen protein combination, should be the striving direction of fmd protein subunit vaccine research.
Used t cell epitope has comprised the conservative t cell epitope on structural protein VP4 and Nonstructural Protein 3A and the 3D in this research, and from two general t cell epitopes in the invasion (invasin) of Measles virus and Yersinia, purpose is to induce most animal all can produce stronger cellullar immunologic response, identification to t cell epitope is limited by the diversity of host expresses MHC molecule, thereby selects to have used the t cell epitope of versatility when design T cellular immunization is former.In addition; in this T cellular immunization is former, also introduce two cytotoxic T cell (Tc) epi-positions that are arranged in VP1 albumen; the Tc epi-position is relevant with the memory immunity; thereby; expect that designed T cellular immunization is former and can induce the host to produce strong and persistent protective immunity; the streaming detected result proves that also designed T cellular immunization is former in inducing CD8+T cell proliferation tool to have certain effect.Used two general t cell epitopes comprise one section 16 amino acid whose t cell epitope from the bacteria attack element, and this section has been proved to be a kind of strong immunostimulant [10].General t cell epitope (PADRE) belongs to Th cell epitope family, and be proved and can have induced multiple animal such as rat, cavy, pigs etc. produce stronger cellullar immunologic response [11-13].Produce strong cellullar immunologic response by inducing, to promote generation at FMDV specificity neutralizing antibody.The immunity of FMDV is typical Cell Dependent Humoral Immune; the immanoprotection action that simple B cell epitope antigen induction body produces is very limited, thereby the introducing of versatility t cell epitope has important effect [13] to the level that improves cellullar immunologic response.
This result of study shows, former foot and mouth disease conventional vaccine and the protein subunit vaccine immune mouse of can further promoting of designed T cellular immunization produces higher levels of neutralizing antibody, this effect induces the high-level cellular immunization of generation to be proportionate with it, illustrate and add the former immune effect that can effectively improve aftosa vaccine of T cellular immunization, be expected to be used for the research of hoof-and-mouth disease subunit vaccine and efficient inactivation vaccine, improve the immune effect of recombinant vaccine and inactivated vaccine.
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Sequence table
Claims (3)
1. a T cellular immunization protogene TI is characterized in that it has the nucleotide sequence of SEQ ID NO:1.
2. T cellular immunization protogene TI according to claim 1 is characterized in that it has the aminoacid sequence of SEQ ID NO:2.
3.T cellular immunization protogene TI uses in fmd protein subunit vaccine and inactivated vaccine.
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