CN110269933A

CN110269933A - A kind of preparation method and applications of rabies viruses subunit vaccine

Info

Publication number: CN110269933A
Application number: CN201910643374.8A
Authority: CN
Inventors: 曹文龙; 孔迪; 滕小锘; 易小萍; 张大鹤
Original assignee: Suzhou Shino Biotechnology Co Ltd
Current assignee: Suzhou Shino Biotechnology Co Ltd
Priority date: 2019-07-17
Filing date: 2019-07-17
Publication date: 2019-09-24

Abstract

The invention discloses a kind of preparation method and applications of rabies viruses subunit vaccine, the vaccine includes: using the nucleic acid molecules of SEQ ID NO:1 or the rabies virus G protein with the identical nucleic acid molecule encoding of 95% or more nucleotide sequence of SEQ ID NO:1.The vaccine uses eukaryotic expression; antigenicity, the immunogenicity of product are similar to native protein; expression is higher; immunogenicity is strong; protecting effect is good; there is no pathogenic and of the invention vaccine that can prepare by the extensive serum free suspension culture of bioreactor to animal, while greatly reducing production of vaccine cost.

Description

A kind of preparation method and applications of rabies viruses subunit vaccine

Technical field

The invention belongs to animal immune technical field of pharmaceuticals more particularly to a kind of preparation sides of rabies viruses subunit vaccine Method and its application.

Background technique

Rabies are infectious diseases that is a kind of ancient and lacking the measure that effectively prevents, the whole world every year there are about 55,000 people die of it is mad Dog disease.Rabies are caused by the central nervous system that rabies viruses infects resurrectionist leads to encephalomyelitis, once morbidity has 100% death rate.According to this rabic feature, world health organisation recommendations are used soaks including vaccine immunity and immune serum The post-exposure prophylaxis (PEP) of profit injection carrys out antirabic generation.Epidemiological survey shows almost all of warm-blooded animal It is all susceptible to hydrophobin.Wild animal is the natural reservoir of hydrophobin, and the domestic animals such as dog, cat are rabies Major transmitter.Therefore, the key of prevention and control human rabies should control animal rabies from source.

Hydrophobin (Rabies Virus, RV) belongs to Rhabdoviridae lyssavirus, and genome is single-stranded The strand RNA of non-segmented negative, from 3 ' to 5 ' are separately encoded N protein, P albumen, M albumen, G-protein and L albumen.G-protein is virus The sole protein of coating forms, and is the principal element for determining that RV is pathogenic, and unique stimulation body generates neutralizing antibody Antigen, and it is related with the budding of virus.

Vaccine inoculation is the rabic unique effective means of control.Currently, prevention rabies are most widely used in the world It is the rabies vacciness of inactivation.But this kind of vaccine needs to be inoculated with spininess just and can induce enough immunoprotections, and expensive. Cost is relatively low for the inactivated vaccine prepared with the nerve fiber of infection animal, but this kind of vaccine can generate serious side reaction, answer With being extremely restricted.Although attenuated live vaccine can effectively inducing protective immunity react, attenuated live vaccine strain sometimes may be used Pathogenicity variation is generated during breeding in vivo due to itself remaining virulence or virus and is caused in the animal of inoculation mad Dog disease, while developing that the attenuated live vaccine period is long, and unpredictability is more.

The present invention is therefore.

Summary of the invention

The present invention overcomes the deficiencies in the prior art, a kind of preparation method of rabies viruses subunit vaccine is provided and its is answered With to solve problems of the prior art.

In order to achieve the above objectives, the technical solution adopted by the present invention are as follows: a kind of immune composition includes:

Using sequence nucleic acid molecules as shown in SEQ ID NO:1 or with nucleotide sequence shown in SEQ ID NO:1 The rabies virus G protein of 95% or more identical nucleic acid molecule encoding.

In a preferred embodiment of the present invention, the rabies virus G protein include SEQ ID NO:2 amino acid sequence or The identical amino acid sequence of 95% or more full length amino acid sequence of person and SEQ ID NO:2.

The present invention also provides a kind of immune compositions to be directed to rabies viruses for inducing in animal subject for producing The purposes of the medicament of the immune response of antigen.

It is used to produce the medicament for preventing animal by rabies virus infection the present invention also provides a kind of immune composition Purposes.

Another object of the present invention is to provide a kind of preparation methods of immune composition, comprising the following steps:

S1, the gene cloning for encoding the rabies virus G protein after codon optimization are contained into carrier for expression of eukaryon There is the recombinant plasmid of rabies virus G protein encoding gene；

S2, by Transfected Recombinant Plasmid Chinese hamster ovary celI described in step S1, obtain Recombinant CHO cell line；

S3, by Recombinant CHO cell line described in culture, screening, domestication step S2, obtain stable capable of highly expressing The Recombinant CHO cell line of G-protein；

S4, fermented and cultured is carried out to the Recombinant CHO cell line in S3 step, obtains recombinant rabies poison G after purification Albumen.

The present invention also provides a kind of preparation methods of immune composition for producing in animal subject Purposes of the induction for the medicament of the immune response of rabies virus antigen.

The present invention also provides a kind of preparation methods of immune composition for producing for preventing animal by mad The purposes of the medicament of dog virus infection.

The present invention also provides a kind of albumen comprising the group being made up of:

The amino acid sequence of SEQ ID NO:2 is identical as 95% or more the full length amino acid sequence of SEQ ID NO:2 Amino acid sequence.

Another object of the present invention is to provide a kind of albumen for producing for preventing animal by rabies viruses The purposes of the medicament of infection.

Another object of the present invention is to provide a kind of albumen to examine for producing for detecting rabies viruses correlation The purposes of disconnected reagent.

The invention solves the defect existing in the background technology, the present invention have it is following the utility model has the advantages that

The invention discloses a kind of preparation method of the rabies viruses genetic engineering subunit vaccine of expressing cho cell and answer With the vaccine can generate stronger humoral immunity in animal body, and stronger more fully antibody can be excited to protect；Structure of the present invention The Recombinant CHO cell line of suspending stabilized efficient secretory expression recombinant rabies poison G-protein is built and has screened, which expresses G Protein yield is high, not only shortens the protein purification time and simplifies production of vaccine step, has been greatly reduced production of vaccine cost； Recombinant rabies poison G-protein production process of the invention is not related to totivirus, has natural safety；The carrier that the present invention uses There is glutamine synthelase (Glutamine Synthetase, GS) to screen amplification system, target gene copy number can be increased, It is expressed, is secreted into supernatant by Chinese hamster ovary celI, be easy to purify, mammalian cell eukaryotic expression is expressed glycosylation modified Perfect, closest to protein molecular itself, suspend culture, is easy to be mass produced；Rabies viruses subunit vaccine of the invention Antigenicity, immunogenicity and function are similar to native protein, and expression is higher, and immunogenicity is strong, does not cause a disease to animal Property, and vaccine of the invention can be prepared by the extensive serum free suspension culture of bioreactor, greatly reduce vaccine Production cost.

Detailed description of the invention

Present invention will be further explained below with reference to the attached drawings and examples；

Fig. 1 is that PCR product carries out gel electrophoresis result after single G-protein target gene carries out PCR amplification, attached in 1.4kbp Purpose band occurs in close position: 1 is purpose gene, and 2 be negative control, M Marker；

Fig. 2 carries out gel electrophoresis for PCR product after the PCR amplification of the bacterium colony sample of multiple G-protein target gene conversion As a result, positive sample nearby occurs in 1.4kbp band: 1 to 5 is the production after the bacterium colony sample P CR amplification of G-protein genetic transformation Object, 6 be negative control；M is Marker；

Fig. 3 is to build the transfer vector pCI-G-GS map containing target gene；

Fig. 4 is the cell culture supernatant progress PAGE gel electrophoresis detection harvested in embodiment 3 as a result, G egg There is purpose band near molecular weight about 55kDa in white cell culture；Wherein 1 cell culture to be harvested in embodiment 3 Object supernatant, 2 be negative control, and M is molecular weight marker；

Fig. 5 is the product Western Blot testing result in embodiment 4 after SDS-PAGE electrophoresis；Wherein 1 is recombination CHO Cell expresses sample, and 2 be negative control, and M is molecular weight marker.

Specific embodiment

Presently in connection with drawings and examples, the present invention is described in further detail, and the embodiment of the present invention is only used for Bright technical solution of the present invention, and the non-limiting present invention.

It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention The identical meanings of understanding.

G-protein sequence can be original series in the present invention, increase and truncated sequence, may include using carrier but It is not limited to pSV2-GS, pCI-GS, pcDNA4-GS, preferably uses pCI-GS.CHO cell line can be as DG44, DXB11, CHO-K1, CHO-S cell strain, preferred CHO-S, but not limited to this.Adjuvant preferably be selected from MONTANIDE ISA 15, One or more kinds of combinations of MONTANIDE ISA 206VG, MONTANIDE GEL 01.

Embodiment 1: the building of recombinant eukaryon expression vector pCI-G-GS

1.1, the building of pUC-G plasmid vector

The RV-G gene of codon optimization comes from Nanjing Genscript Biotechnology Co., Ltd., and is cloned into pUC-57 carrier On, construct pUC-G plasmid vector.G gene order after optimization is as shown in SEQ ID NO:1.

1.2, G gene magnification

Using pUC-G as template, G-F, G-R carry out the PCR amplification (gene order of G-F such as SEQ ID NO.3 as primer Shown, the gene order of G-R is as shown in SEQ ID NO.4), amplification system is shown in Table 1.Reaction condition are as follows: 94 DEG C of initial denaturations 5 are divided Clock；95 DEG C be denaturalized 45 seconds, 60 DEG C renaturation 45 seconds, 72 DEG C extend 2 minutes, 30 circulation；72 DEG C extend 10 minutes, 4 DEG C of preservations.

1 G gene magnification system of table

PCR product is subjected to gel electrophoresis and identifies target gene size, as shown in Figure 1, item occurs in the position in 1.4kbp Band, target gene expand successfully, carry out recovery purifying with gel purification kit.

1.3, digestion

G gene PCR product by pCI-GS plasmid and after purification uses Xho I, I 37 DEG C of Kpn digestion 3 hours respectively, reaction System is shown in Table 2, table 3.It is separately recovered after digestion products gel electrophoresis, is purified with gel purification kit.

2 G gene endonuclease reaction system of table

3 pCI-GS plasmid enzyme restriction reaction system of table

1.4, it connects

The pCI-GS plasmid of digestion is connected overnight with G gene digestion products using the 16 DEG C of water-baths of T4DNA ligase, even Junctor system is shown in Table 4.

4 G gene of table and pCI-GS plasmid linked system

1.5, it converts

Take 10 μ l connection products be added 100 μ l DH5 α competent cell, mix, 42 DEG C heat shock 90 seconds, ice bath 2 divides The LB culture medium that 900 μ l are free of Amp is added in clock, and 37 DEG C are cultivated 1 hour.1.0ml bacterium solution centrifugal concentrating is taken to be coated at 100 μ l On LB solid medium containing Amp, 37 DEG C are cultivated 16 hours.

1.6, bacterium colony PCR and sequencing identification

Single colonie on picking plate is inoculated with LB liquid medium respectively, and 37 DEG C are cultivated 2 hours, using bacterium solution as template, G-F and G-R carries out bacterium colony PCR as primer.PCR product is subjected to gel electrophoresis verifying purpose gene size, as shown in Fig. 2, The sample for 1.4kbp band occur is positive sample.Sequencing company is sent to be sequenced the positive bacterium solution of bacterium colony PCR identification, selection sequencing Correct bacterium solution is saved, and carrier for expression of eukaryon pCI-G-GS is obtained.The Vector map built is as shown in Figure 3.

Embodiment 2: the building and screening of recombinaant CHO cell

1. cell transfecting

1.1, prepare the Chinese hamster ovary celI of cell logarithmic growth phase, CDG44 cell or DXB11 cell can be used in Chinese hamster ovary celI Or CHO-K1 cell or CHO-S cell, CHO-S cell is selected in the present embodiment, and sampling counts, with 1 × 10⁶Cells/ml's is thin Born of the same parents' density continues to pass on, and maintains seed, and remaining cell centrifugation after 1000rpm is centrifuged 4 minutes, abandons supernatant, new with 20ml or so Fresh CHO-WM culture medium is resuspended, and is centrifuged again, and 1000rpm is centrifuged 4 minutes, abandons after supernatant and counting is resuspended with a small amount of culture medium, most Cell density is adjusted to 1.43 × 10 at last⁷cells/ml。

1.2, the resulting 5 μ g of pCI-G-GS plasmid vector of step 1.6 in plasmid and mixing with cells Example 1 is added extremely In EP pipe, 0.7ml cell is added, after mixing, stands 15 minutes.

1.3 electricity turn 280V 20ms and shock by electricity 2 pulses, after the completion of electric shock, cell are transferred into shaking flask at once, suspend training It supports, cell state is observed after 48h, changes liquid culture, cell densities is waited to grow into 0.6 × 10⁶When cells/ml, 50 μM of MSX are added (L-methionine sulphoximine) pressurization screening.

2. monoclonal screens

2.1, it is trained with the Chinese hamster ovary celI Serum-free and protein-free medium CHO-WM cell of Suzhou Wo Mei Bioisystech Co., Ltd + 50 μM of MSX of base suspension cell again is supported, is counted.

2.2, to 5/mL, the cell for taking 200 μ l to mix is added in 96 orifice plates bed board diluting cells, is placed into 37 DEG C, 5%CO₂4-6h is incubated in cell incubator.Record the hole of individual cells.

2.3, when the hole length of individual cells in 96 orifice plates is got up, culture medium is discarded, PBS is washed once, 100 μ l 0.25% Trypsin-EDTA, room temperature digest 2min or so, and 2mL CHO-WM culture medium (containing 10%FBS+50 μM of MSX) termination is added and disappears Change reaction, and is dispelled cell with pipettor.Cell is transferred to 12 orifice plates, when 12 orifice plates cover with, takes supernatant, Elisa inspection Survey whether clone is the positive, the positive colony of high efficient expression continues to expand culture, freeze.

3. cell shake flask fermentation

3.1, it the configuration of secondary culture base: uses CHO-WM culture medium to add 50 μM of MSX as secondary culture base, is placed in 37 DEG C of water-baths are always preheated to 37 DEG C.

3.2 from CO₂Constant-temperature table takes out shaking flask cell, is counted.

3.3 diluting cells are to 2.5-3.5 × 10⁵A cell/mL inoculation 30mL culture is based in a 125mL shaking flask.Carefully Born of the same parents' culture bottle is placed into 37 DEG C, 5%CO₂100rpm/min is incubated overnight in constant-temperature table.

3.4, every counting cell density and vigor for 24 hours, survey glucose, when sugar is lower than 2g/L, add glucose To 4g/L；1mL sample is taken daily, and supernatant is for detecting protein expression situation.

Embodiment 3:SDS-PAGE detection

The cell culture supernatant harvested in embodiment 2 is subjected to SDS-PAGE detection, while being made using empty Chinese hamster ovary celI For negative control.Concrete operations are as follows: the cell culture for taking 40 μ l to harvest, and 5 × load buffer of 10 μ l is added (loading buffer), boiling water bath 5 minutes, 12000r/min was centrifuged 1 minute, and supernatant is taken to carry out PAGE gel (12% Concentration gel) electrophoresis, take gel to be dyed after electrophoresis, decolourize after observe purpose band.The testing result of culture in embodiment 2 As shown in figure 4, occurring purpose band near molecular weight about 55kDa, negative control does not have band in corresponding position.

Embodiment 4:Western Blot detection

Product after SDS-PAGE electrophoresis in embodiment 3 is transferred on NC (nitrocellulose) film, with 5% skim milk Closing 2 hours, the anti-RV polyvalent antibody of source of mouse are incubated for 2 hours, and rinsing, it is small that the sheep anti mouse polyclonal antibody secondary antibody of HRP label is incubated for 2 When, then rinsing is added dropwise enhanced chemical luminous fluorescent substrate, is taken pictures using chemiluminescence imaging instrument.As shown in figure 5, in figure Recombinant C HO Supernatant samples have cell band, and negative control does not have purpose band, and illustration purpose antigen protein is in recombinaant CHO cell In correctly expressed.

Embodiment 5: the expression of rabies virus G protein is quantitative and fine jade expands bioactivity

The above-mentioned recombinant rabies poison G-protein content of preparation is detected using Elisa method.Mode of operation is as follows: with coating Buffer dilutes rabies viruses virus polyvalent antibody to suitable concentration, and every 100 μ l of hole, 4 DEG C are overnight, and PBST is washed three times, and 1% BSA closes 1h.Be added various concentration antigen standard (chromatographed by exchange of particles, hydrophobic chromatography, what molecular sieve purification obtained Albumen) and gradient dilution measuring samples, 37 DEG C are incubated for 1 hour, and PBST is washed three times.Detection antibody-G-protein monoclonal is added in every hole Antibody, 37 DEG C are incubated for 1 hour, and PBST is washed three times.The sheep anti-mouse igg of two anti-i.e. HRP labels is added in every hole, and 37 DEG C of incubations 1 are small When, PBST is washed three times.TMB develops the color 10 minutes, 2M H₂SO₄Terminate reaction.Microplate reader reading, is calculated by standard curve to sample The amount of recombinant rabies poison G-protein in product.

Elisa testing result is as follows, the average content of G-protein about 167mg/L respectively in stoste.

Using the G-protein potency of fine jade expanding method detection expression, plum blossom hole is beaten on agarose gel plate, among plum blossom hole Be added RV fine jade expand examination criteria serum, be around separately added into dilute 20,1,2,3,4,5,6,7,8,9,10 powers expression Antigen.It is inverted after being incubated for 72h and observes precipitation line.The maximum dilution ratio for precipitation line occur is that its fine jade expands potency.Fine jade expands potency inspection It is as follows to survey result: it is 1:256 that RV-G albumen fine jade, which expands potency,.

Embodiment 6: subunit vaccine preparation

The expression antigen supernatant of harvest is used into normal saline dilution, so as to reach 80 μ g/mL right for the concentration of RV-G albumen The recombinant rabies poison G-protein of purifying is added in MONTANIDE ISA 206VG adjuvant (volume ratio 1:1) afterwards, is emulsified, Quality inspection qualification is placed on 4 DEG C of preservations.

Embodiment 7: immunization experiment

Vaccine in embodiment 6 is randomly selected 5, vaccine sample to be checked is used as after mixing.Quarantine seedling and mad is treated respectively Dog disease inactivated vaccine standard items (the NIH potency of standard vaccine, which is subject to, indicates potency) carry out 5 times and are serially diluted, and take 4 respectively (standard vaccine takes 25,125,625 and 3125 this 4 dilutions to acceptable diluent degree, and vaccine to be checked takes 5,25,125 and 625 This 4 dilutions).13-16gSPF of weight mouse 20, every 0.5mL is injected intraperitoneally in each dilution.Head exempts from 7 days latter, use Head exempt from same batch, same to dilution, with dose vaccine booster immunization 1 time.Head exempts from 14 days afterwards, attacks poison to all mouse intracerebral injections With CVS-24 plants of Mice brain tissues viral suspensions of hydrophobin, every 0.03mL is (containing about 50LD₅₀).It attacks after poison in 4 days, every group In dead mouse should be no more than 4.Record is attacked after poison in 5-14 days in each group because of the mouse quantity of rabies death, calculating The effect (IU/mL) of vaccine to be checked is calculated according to the following formula in each group survival rate.The NIH potency of vaccine to be checked is not less than 2.2IU/ mL.The HIN potency of vaccine to be checked should be 10.72IU/ml, be higher than national standard 2.0IU/ml, illustrate product qualification.

Vaccine NIH potency (IU/mL) to be checked=standard vaccine effect x 5^{(the sum of the sum of vaccine each group survival ratio to be checked-standard vaccine each group survival ratio -1)}

Immunization experiment result is as shown in the table:

Based on the above description of the preferred embodiments of the present invention, through the above description, related personnel completely can be with Without departing from the scope of the technological thought of the present invention', various changes and amendments are carried out.The technical scope of this invention It is not limited to the contents of the specification, it is necessary to determine the technical scope according to the scope of the claims.

Sequence table

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<120>a kind of preparation method and applications of rabies viruses subunit vaccine

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atcgatctgc accatctgtc ttgccccaac aatctggtgg tggaggatga gggctgtaca 180

aacctgagcg gcttttctta catggagctg aaggtcggct atatctccgc catcaaggtg 240

aacggcttca catgcaccgg cgtggtgacc gaggctgaga catacaccaa ttttgtgggc 300

tatgtgacca caaccttcaa gaggaagcac tttcggccaa caccagacgc ttgtagggct 360

gcttacaact ggaagatggc tggcgatcct cggtatgagg agtctctgca caatccttac 420

ccagactatc attggctgag aaccgtgaag acaaccaagg agtccctggt catcatctcc 480

cccagcgtga cagacctgga cccctacgac aagtccctgc atagccgcgt gttcccagga 540

ggaaactgct ccggcatcac agtgtccagc acctactgta gcacaaacca cgattatacc 600

atctggatgc ctgagaatct gaggctgggc acctcctgcg acatcttcac acactccagg 660

ggcaagaggg cctccaaggg cgacaagaca tgtggctttg tggatgagag gggcctgtat 720

aagagcctga agggcgcctg caagctgaag ctgtgcggcg tgctgggact gaggctgatg 780

gacggcacct gggtggctat gcagacctcc gatgagacaa agtggtgccc ccctggccag 840

ctggtgaatc tgcacgactt cagaagcgat gagatcgagc atctggtgga ggaggagctg 900

gtgaagaagc gcgaggagtg tctggatgcc ctggagagca tcatgacaac caagtccgtg 960

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atctttaaca agacactgat ggaggccgac gctcattata agtctgtgca gacctggaat 1080

gagatcatcc catccaaggg ctgcctgaga gtgggagaga ggtgccaccc acatgtgaac 1140

ggcgtgttct ttaatggcat catcctgggc tccgatggcc acgtgctgat cccagagatg 1200

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catcccctgg ctgatcctag caccgtgttc aaggacggcg atgaggtgga ggactttgtg 1320

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Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro

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Trp Ser Pro Ile Asp Leu His His Leu Ser Cys Pro Asn Asn Leu Val

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Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu

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Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys

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Thr Gly Val Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr

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Val Thr Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala

100 105 110

Cys Arg Ala Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu

115 120 125

Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val

130 135 140

Lys Thr Thr Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Thr Asp

145 150 155 160

Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Gly Gly

165 170 175

Asn Cys Ser Gly Ile Thr Val Ser Ser Thr Tyr Cys Ser Thr Asn His

180 185 190

Asp Tyr Thr Ile Trp Met Pro Glu Asn Leu Arg Leu Gly Thr Ser Cys

195 200 205

Asp Ile Phe Thr His Ser Arg Gly Lys Arg Ala Ser Lys Gly Asp Lys

210 215 220

Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly

225 230 235 240

Ala Cys Lys Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp

245 250 255

Gly Thr Trp Val Ala Met Gln Thr Ser Asp Glu Thr Lys Trp Cys Pro

260 265 270

Pro Gly Gln Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu

275 280 285

His Leu Val Glu Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp

290 295 300

Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu

305 310 315 320

Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile

325 330 335

Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Gln

340 345 350

Thr Trp Asn Glu Ile Ile Pro Ser Lys Gly Cys Leu Arg Val Gly Glu

355 360 365

Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu

370 375 380

Gly Ser Asp Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu

385 390 395 400

Gln Gln His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Met His

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Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Val Glu

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Claims

1. a kind of immune composition, it is characterised in that: include:

Using sequence nucleic acid molecules as shown in SEQ ID NO:1 or with nucleotide sequence 95% shown in SEQ ID NO:1 The rabies virus G protein of the above identical nucleic acid molecule encoding.

2. a kind of immune composition according to claim 1, which is characterized in that the rabies virus G protein includes SEQ The amino acid sequence of ID NO:2 or the identical amino acid sequence of 95% or more full length amino acid sequence with SEQ ID NO:2.

3. immune composition according to claim 1 or 2 is directed to rabies for inducing in animal subject for producing The purposes of the medicament of the immune response of malicious antigen.

4. immune composition according to claim 1 or 2 is used to produce the medicine for preventing animal by rabies virus infection The purposes of agent.

5. a kind of preparation method of immune composition, which comprises the following steps:

S1, the gene cloning for encoding the rabies virus G protein after codon optimization are obtained into carrier for expression of eukaryon containing mad The recombinant plasmid of dog viral G protein encoding gene；

S3, by Recombinant CHO cell line described in culture, screening, domestication step S2, obtain stable capable of highly expressing G egg White Recombinant CHO cell line；

S4, fermented and cultured is carried out to the Recombinant CHO cell line in S3 step, obtains recombinant rabies poison G-protein after purification.

6. a kind of preparation method of immune composition according to claim 5 is for producing for inducing in animal subject For the purposes of the medicament of the immune response of rabies virus antigen.

7. a kind of preparation method of immune composition according to claim 5 is for producing for preventing animal by rabies The purposes of the medicament of poison infection.

8. a kind of albumen comprising the group being made up of:

The amino acid sequence of SEQ ID NO:2 or the identical ammonia of 95% or more full length amino acid sequence with SEQ ID NO:2 Base acid sequence.

9. albumen according to claim 8 is for producing for preventing animal by the purposes of the medicament of rabies virus infection.

10. albumen according to claim 8 is used to produce the purposes for detecting rabies viruses dependent diagnostic reagent.