CN108912213A - The immunogenic polypeptide and the preparation method and application thereof of enterovirns type 71 VP1 antigen - Google Patents

The immunogenic polypeptide and the preparation method and application thereof of enterovirns type 71 VP1 antigen Download PDF

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CN108912213A
CN108912213A CN201810796493.2A CN201810796493A CN108912213A CN 108912213 A CN108912213 A CN 108912213A CN 201810796493 A CN201810796493 A CN 201810796493A CN 108912213 A CN108912213 A CN 108912213A
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immunogenic polypeptide
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CN108912213B (en
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温红玲
郝树彬
马英伟
陶泽新
王志玉
庄志超
白永娟
王莉鸿
李纯
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Shandong University
Shandong Quality Inspection Center for Medical Devices
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Shandong Quality Inspection Center for Medical Devices
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32311—Enterovirus
    • C12N2770/32322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus

Abstract

The invention belongs to field of immunobiology, are related to a kind of immunogenic polypeptide and the preparation method and application thereof of enterovirns type 71 VP1 antigen.Specifically, it is related to a kind of immunogenic polypeptide of enterovirns type 71 VP1 antigen, it has the amino acid sequence as shown in SEQ ID NO.1-SEQ ID NO.5 any sequence in sequence table or above-mentioned sequence through replacement, missing or one or several amino acids formed amino acid sequences with same function of addition.The invention further relates to the preparation methods of the immunogenic polypeptide and the purposes in the diagnostic reagent for preparing EV71 virus.Immunogenic polypeptide of the invention has good antigenicity and immunogenicity, provides Research foundation for the exploitation of early diagnosis reagent and vaccine research and development of EV71.

Description

The immunogenic polypeptide and the preparation method and application thereof of enterovirns type 71 VP1 antigen
Technical field
The invention belongs to immuno-biology technical fields, and in particular to a kind of immunogene of enterovirns type 71 VP1 antigen Property polypeptide and the preparation method and application thereof.
Background technique
EV71 is single strand plus RNA virus, is subordinated to Picornaviridae enterovirus A category, and infection can cause infant Hand-foot-and-mouth disease, when morbidity, are mainly shown as fever, diarrhea and herpangina.It is certain unlike typical hand-foot-and-mouth disease EV71 virus strain infection can cause severe hand-foot-and-mouth disease, complication include aseptic meningitis, encephalitis, polio sample paralysis, Neurogenic pulmonary edema is even dead.EV71 infection morbidity is extensive, and Epidemic Scope involves Asia, Europe, North America, Australia It is high Deng, the death rate, it is current quite valued public health problem.
For many years, scholars have been devoted to the research and development of EV71 vaccine, learn as molecular biology and immunology etc. The cross development of section, recombinant subunit vaccine, DNA vaccination, the virus sample particle vaccines of EV71 are come out one after another.Wherein EV71 tradition Full inactivated vaccine is with the fastest developing speed, III clinical trial phase that current China has three mechanisms that the full inactivated vaccine of EV71 is completed. But full inactivated vaccine is there are problems, such as immune effect are generally lower, can not inducing cytotoxic T lymphocyte reaction, lure The artificial delivery raw immune response duration is shorter, and inactivator has influence etc. to viral antigen；So research and development new generation vaccine is needed, Effective stimulus immune response while retaining intact immune originality, avoids infection simultaneously.
About the Infect And Diagnose of EV71, conventional method includes neutralizing experimental method and isolation of virus, and above method is time-consuming to take Power, requirement of experiment is higher, is unsuitable for clinical application.In recent years, (such as reverse transcriptase polymerase chain formula is anti-for molecular Biological Detection technology Answer (RT-PCR), Fluorescent quantitative PCR (Quantitative Real-time PCR)) for diagnosing EV71 infection using more and more, but there are false positive and false negatives for such method, more demanding to experimental technique, are not easy In Grass-roots Hospital.And the principle that enzyme linked immunosorbent assay (ELISA) and immunochromatographic method (ICA) utilize antigen-antibody to combine The detection of EV71 is carried out, it is easy to operate quick, there are many correlative studys at present, but not yet clinically promote.
EV71 can be divided into tri- hypotypes of A, B, C, and wherein B and C hypotype can be respectively divided into B1-B5 and C1-C5 type again. EV71 An open reading frame (open reading frame, ORF) is contained only, the upstream and downstream of ORF is respectively 5 ' UTR and 3 ' UTR.Gene Group containing about 7500 nucleotide of RNA overall length, encode the polyprotein with 2193 amino acid, and polyprotein can be hydrolyzed to Tri- precursor proteins of P1, P2, P3 are finally hydrolyzed to 4 structural proteins (VP1-VP4) and 7 non-structural proteins (2A-2C, 3A- 3D).In 4 structural proteins, VP4 is embedded in the inside of virus coat, and excess-three is both exposed to surfaces of viral particles, directly connects Touch host immune system；Wherein VP1 albumen has genetic diversity corresponding with serotype, determines comprising the main antigen of EV71 Determine cluster, therefore VP1 is one of the hot spot studied at present, has been widely used in the Pathogen test and vaccine research of hand-foot-and-mouth disease.
The area BC loop (91-106 amino acid sites) of VP1 albumen is neutralization epitope sites generally acknowledged at present, but Whether there is also other important antigenic determinants, it is still necessary to further study demonstration.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide a kind of immunogenicities of enterovirns type 71 VP1 antigen Polypeptide and the preparation method and application thereof.VP1 albumen is split, the antigenicity and immunogenicity of each peptide fragment are studied, is EV71 Early diagnosis reagent and vaccine research and development provide basic research.
To achieve the above object, the present invention adopts the following technical scheme that：
One aspect of the present invention is related to a kind of immunogenic polypeptide of enterovirns type 71 VP1 antigen, has such as sequence In list amino acid sequence shown in SEQ ID NO.1-SEQ ID NO.5 any sequence or above-mentioned sequence through replacement, missing or Add one or several amino acids formed amino acid sequences with same function.
Another aspect of the invention is related to encoding the polynucleotides of above-mentioned immunogenic polypeptide；Specifically, the multicore The sequence of thuja acid is respectively as shown in SEQ ID NO.6-SEQ ID NO.10.
The present invention also provides the recombinant vectors for containing above-mentioned polynucleotide sequence.
Recombinant vector above-mentioned, the carrier that sets out are pET-49b (+).
The present invention also provides the recombinant expression bacterium for containing above-mentioned recombinant vector.
Recombinant expression bacterium above-mentioned, starting strain are Escherichia coli DE3.
The present invention also provides the expression of the immunogenic polypeptide of enterovirns type 71 VP1 antigen and purification process comprising Following steps：
(1) by genetic fragment and carrier pET-49b (+) shown in SEQ ID NO.6-SEQ ID NO.10, through SpeI and After III double digestion of Hand, connection building obtains expression vector pET-49b (+)-VP1, pET-49b (+)-VP179-432、 pET-49b (+)-VP1436-864、pET-49b(+)-VP1436-735With pET-49b (+)-VP1565-864；
(2) by expression vector pET-49b (+)-VP1, pET-49b (+)-VP1 of step (1)79-432、pET-49b(+)- VP1436-864、 pET-49b(+)-VP1436-735With pET-49b (+)-VP1565-864Conversion screens positive gram to Escherichia coli DE3 It is grand, inducing expression recombinant protein；
(3) by the recombinant protein of step (2) using glutathione sulfydryl transferase (GST) agarose gel purification to get.
In step (1), the construction method of genetic fragment shown in SEQ ID NO.6-SEQ ID NO.10 is：With SDLY107 (GenBank:JX244186.1) full-length cDNA is pcr template, shown in SEQ ID NO.11-SEQ ID NO.20 Primer sequence be amplimer, obtain VP1 full length gene segment and 4 sections of VP1 genetic fragments through PCR amplification, respectively VP1, VP179-432、VP1436-864、VP1436-735And VP1565-864。
The primer sequence of amplification is specific as follows：
SpeI VP1 Primer F：5'-CGGACTAGTGGAGATAGGGTGGCAGATGTAATTG-3'；(SEQ ID NO.11)；
HindIII VP1 Primer R:5'-CCCAAGCTTCTAAAGAGTGGTGATCGCTGTGCG-3'；(SEQ ID NO.12)；
1 VP1 SpeI Primer F:5'-CGGACTAGTATGCCCACAGGCCAGAACACACAGGTGA-3'； (SEQ ID NO.13)；
1 VP1 HindIII Primer R:5'-CCCAAGCTTCTACCCGGTGGGTGTGCACGCAACAAAA-3'； (SEQ ID NO.14)；
2 VP1 SpeI Primer F:5'-CGGACTAGTATGGTTGTCCCACAATTGCTCCAATATA-3'； (SEQ ID NO.15)；
2 VP1 HindIII Primer R:5'-CCCAAGCTTCTAACCAGTTGGCTTAATGGAGTTG-3'； (SEQ ID NO.16)；
3 VP1 SpeI Primer F:5'-CGGACTAGTGTTGTCCCACAATTGCTCCAATA-3'；(SEQ ID NO.17)；
3 VP1 HandIII Primer R:5'-CCCAAGCTTCTAGTACTTGGACTTGGAGGTCC-3'；(SEQ ID NO.18)；
4 VP1 SpeI Primer F:5'-CGGACTAGTCAGGTTTCAGTGCCATTCATGTC-3'；(SEQ ID NO.19)；
4 VP1 HandIII Primer R:5'-CCCAAGCTTCTAACCAGTTGGCTTAATGGAGTT-3'；(SEQ ID NO.20)。
The present invention also provides the immunogenic polypeptides of the VP1 antigen of above-mentioned enterovirns type 71 to prepare examining for EV71 virus Application in disconnected reagent.
Beneficial effects of the present invention：
(1) present invention has found except the area BC loop (91-106 amino acid sites) new on EV71 Structural protein VP1 Immunogenic polypeptide, the polypeptide have good antigenicity and immunogenicity, the early diagnosis reagent exploitation for being EV71 and vaccine Research and development provide Research foundation.
(2) expression of immunogenic polypeptide of the invention and purification process are simple, convenient, time saving, are advantageously implemented Large-scale industrial production.
Detailed description of the invention
Fig. 1：The expression of VP1 albumen and qualification result；In figure, 1：Albumen marker；2：The unconverted DE3 bacterium for crossing plasmid Body；3：It is the IPTG inducing expression 3h of 0.33mmol/L with concentration；4：It is the IPTG inducing expression 3h of 0.67mmol/L with concentration； 5：It is the IPTG inducing expression 4h of 0.33mmol/L with concentration；6：It is the IPTG inducing expression 4h of 0.67mmol/L with concentration；7： It is the IPTG inducing expression 5h of 0.33mmol/L with concentration；8：It is the IPTG inducing expression 5h of 0.67mmol/L with concentration.VP1- Gst fusion protein molecular weight is 59kd.
Fig. 2：Immunogenic polypeptide VP179-432Expression and qualification result；In figure, 1：Albumen marker；2：It is with concentration The IPTG inducing expression 3h of 0.33mmol/L；3：It is the IPTG inducing expression 3h of 0.67mmol/L with concentration；4：It is with concentration The IPTG inducing expression 4h of 0.33mmol/L；5：It is the IPTG inducing expression 4h of 0.67mmol/L with concentration；6：It is with concentration The IPTG inducing expression 5h of 0.33mmol/L；7：It is the IPTG inducing expression 5h of 0.67mmol/L with concentration. VP179-432-GST Fusion protein molecule amount is about 39kd；8：The unconverted DE3 thallus for crossing plasmid.
Fig. 3：Immunogenic polypeptide VP1436-864Expression and qualification result；In figure, 1：Albumen marker；2：It is with concentration The IPTG inducing expression 3h of 0.33mmol/L；3：It is the IPTG inducing expression 3h of 0.67mmol/L with concentration；4：It is with concentration The IPTG inducing expression 4h of 0.33mmol/L；5：It is the IPTG inducing expression 4h of 0.67mmol/L with concentration；6：It is with concentration The IPTG inducing expression 5h of 0.33mmol/L；7：It is the IPTG inducing expression 5h of 0.67mmol/L with concentration. VP1436-864GST Fusion protein molecule amount is about 40kd；8：The unconverted DE3 thallus for crossing plasmid.
Fig. 4：Immunogenic polypeptide VP1436-735Expression and qualification result；In figure, 1：Albumen marker；2：Unconverted mistake The DE3 thallus of plasmid；3：It is the IPTG inducing expression 3h of 0.33mmol/L with concentration；4：It is 0.67mmol/L's with concentration IPTG inducing expression 3h；5：It is the IPTG inducing expression 4h of 0.33mmol/L with concentration；6：It is 0.67mmol/L's with concentration IPTG inducing expression 4h；7：It is the IPTG inducing expression 5h of 0.33mmol/L with concentration；8：It is 0.67mmol/L's with concentration IPTG inducing expression 5h.VP1436-735Gst fusion protein molecular weight is 40kd.
Fig. 5：Immunogenic polypeptide VP1565-864Expression and qualification result；In figure, 1：Albumen marker；2：Unconverted mistake The DE3 thallus of plasmid；3：It is the IPTG inducing expression 3h of 0.33mmol/L with concentration；4：It is 0.67mmol/L's with concentration IPTG inducing expression 3h；5：It is the IPTG inducing expression 4h of 0.33mmol/L with concentration；6：It is 0.67mmol/L's with concentration IPTG inducing expression 4h；7：It is the IPTG inducing expression 5h of 0.33mmol/L with concentration；8：It is 0.67mmol/L's with concentration IPTG inducing expression 5h.VP1565-864Gst fusion protein molecular weight is 37kd.
Fig. 6：The VP1 protein antigenicity testing result of purifying；In figure, 1：Negative control；2：VP1-GST fusion after purification Albumen, VP1-GST fusion protein molecule amount are 59kd；3：Unpurified VP1-GST fusion protein；Primary antibody is the anti-EV71 of source of mouse Serum.
Fig. 7：The VP1 of purifying79-432Protein antigenicity testing result；In figure, 1：Unpurified VP179-432- GST merges egg It is white, VP179-432The molecular weight of gst fusion protein is about 39kd；2-3：The VP1 of purifying79-432Gst fusion protein；4：It is negative right According to；Primary antibody is the anti-EV71 serum of source of mouse.
Fig. 8：The VP1 of purifying436-864Protein antigenicity testing result；In figure, 1：Unpurified VP1436-864- GST merges egg It is white, VP1436-864The molecular weight of gst fusion protein is about 40kd；2：The VP1 of purifying436-864Gst fusion protein；3：It is negative right According to；Primary antibody is the anti-EV71 serum of source of mouse.
Fig. 9：The VP1 of purifying436-735Protein antigenicity testing result；In figure, 1：Unpurified VP1436-735- GST merges egg It is white, VP1436-735The molecular weight of gst fusion protein is about 40kd；2：The VP1 of purifying436-735- GST albumen；3：Negative control；One Resist the anti-EV71 serum for source of mouse.
Figure 10：The VP1 of purifying565-864Protein antigenicity testing result；In figure, 1：Unpurified VP1565-864- GST fusion Albumen, VP1565-864The molecular weight of gst fusion protein is about 37kd；2：The VP1 of purifying565-864- GST albumen；3：Negative control； Primary antibody is the anti-EV71 serum of source of mouse.
Figure 11：The qualification result of the immunogenicity of VP1 albumen and immunogenic polypeptide；In figure, 1,2,3 be respectively EV71 Virus, negative control and albumen marker.Negative control is supernatant after the centrifugation of RD cell.
Specific embodiment
The present invention is further illustrated in conjunction with the embodiments, it should which explanation, following the description is merely to explain this Invention, is not defined its content.
Used experimental material and reagent are as follows in following embodiments：
1. experimental material：
1.1 cells, virus and experimental animal
Cell used is people's human rhabdomyosarcoma cells (rhabdomyosarcoma cells, RD).RD cell is grown to pair Inoculation EV71SDLY107 virus when number growth period, when cytopathy is to 80% or so, multigelation between -40 DEG C and room temperature 4 DEG C of 10,000r/min, which are centrifuged, after three times takes supernatant for 10 minutes, and harvest virus is simultaneously stored in -80 DEG C of refrigerators.Balb/c mouse Totally 25, Kunming mouse 5, it is purchased from Shandong University's Experimental Animal Center.
1.2 experiment reagent
Main agents used are virus RNA extraction kit (OMEGA, China), Reverse Transcriptase kit (TOYOBO, day Originally), DNA plastic recovery kit and plasmid extraction kit (OMEGA, China)；Cell culture medium, serum (HyClone, beauty State)；LA Taq enzyme, T4DNA ligase, DNA Marker (Takara, China)；Restriction enzyme, albumen Marker (Thermo, the U.S.)；Pvdf membrane (Millipore, the U.S.)；5 × albumen sample-loading buffer (the green skies, China)；BCA albumen Quantification kit (Ai Delai, China)；Horseradish enzyme marks mountain sheep anti-mouse igg, concentrated type DAB kit (Zhong Shan Golden Bridge, China)； GST monoclonal antibody (Proteintech, the U.S.)；TMB one-component developing solution (Suo Laibao, China)；GST Ago-Gel (health is century, China)；Primer is synthesized by Shanghai Sheng Gong bioengineering limited liability company；Sequencing wins still biotechnology by Shanghai Company completes.
Embodiment 1：The expression and purifying of the immunogenic polypeptide of the VP1 antigen of enterovirns type 71
1. expanding the cDNA of EV71 SDLY107 strain virus
The RNA of SDLY107 strain virus, Strain are extracted using the Viral RNA kit kit of OMEGA company SDLY107 is isolated from the hand-foot-and-mouth disease infant of People's Hospital of Linyi City, Shandong Province death, and being identified as velogen strain, (virus strain infection is thin After born of the same parents, cytopathy becomes faster and strong, and animal pathological characters are obvious), strain has carried out complete sequence analysis (GenBank: JX244186.1), cDNA is generated using the ReverTra Ace qPCR Kit reverse transcription of TOYOBO company.
2. design of primers and PCR amplification
Using SDLY107 full-length cDNA as pcr template, the primer of PCR amplification is designed, primer sequence is as shown in table 1：
1 primer sequence of table
Using SDLY107 full-length cDNA as pcr template, SpeI VP1 Primer F is upstream primer, HindIII VP1 Primer R is the VP1 full-length gene that downstream primer expands SDLY107 strain (as shown in SEQ ID NO.6)；With 1 VP1 SpeI Primer F/1 VP1 HindIII Primer R、2 VP1 SpeI Primer F/2 VP1 HindIII Primer R, 3 VP1 SpeI Primer F/3 VP1 HindIII Primer R and 4VP1 SpeI Primer F/4VP1 HindIII Primer R is 4 sections of VP1 genetic fragments of primer amplification, i.e.,：VP179-432(as shown in SEQ ID NO.7), VP1436-864(such as Shown in SEQ ID NO.8), VP1436-735(as shown in SEQ ID NO.9), VP1565-864(as shown in SEQ ID NO.10).
PCR condition：94 DEG C of initial denaturation 10min；94 DEG C of denaturation 30s；55 DEG C of annealing 30s；72 DEG C of extension 1min；72 DEG C of terminals Extend 10min totally 30 circulations.
3. the building of expression vector
Gained DNA piece in SpeI and HindIII double digestion pET-49b (+) carrier and " 2. design of primers and PCR amplification " Section：SpeI and each 2 μ L of HindIII FastDigest enzyme, 10 μ g, 10 × FastDigest Green of DNA fragmentation 5 μ l of Buffer, water polishing to 50 μ l, 37 DEG C of digestion 4h.Glue recycles endonuclease bamhi, and the connection of T4 ligase obtains recombination matter overnight Grain pET-49b (+)-VP1, pET-49b (+)-VP179-432、pET-49b(+)-VP1436-864、pET-49b(+)-VP1436-735、 pET-49b(+)-VP1565-864。
The expression and identification of 4.VP1 albumen and immunogenic polypeptide
Above-mentioned constructed 10 μ L of recombinant plasmid is converted into 100 μ L Escherichia coli DE3, LB liquid medium is added After 890 μ L, 37 DEG C of 160rpm 1h, 100 μ L bacterium solutions are taken to be spread evenly across solid agar medium, 37 DEG C overnight.Next day picking warp Kanamycins (30 μ g/ml) screens positive bacterium colony, is inoculated in 5mL LB (containing kanamycins) fluid nutrient medium, 37 DEG C 160rpm shake culture is stayed overnight；Next day in proportion 1:100 are inoculated in fresh LB liquid medium, 37 DEG C of shake cultures to OD When value reaches 0.8-1.0, isopropyl-β-D Thiogalactopyranoside (IPTG) 0.33mmol/L is added, continues to cultivate 4-5h, 1mL bacterium solution is taken, 4 DEG C of 10000r/min centrifugation 2min collect thallus, 80 μ L phosphate buffers (PBS) are added, thallus is resuspended, Above-mentioned sample presses 1：4 are added 5 × SDS-PAGE sample-loading buffer, boiling water bath 7min after mixing, 4 DEG C of 10000r/min centrifugations 2min, after taking supernatant to carry out electrophoresis, by bacterium solution protein delivery to nitrocellulose filter, 5% skimmed milk power closes 40min；One The anti-anti-GST antibody (1 with rabbit source:5000) 4 DEG C of overnight incubations, after TBST washes film 3 times, horseradish peroxidase mark is added in secondary antibody The goat anti-rabbit IgG antibody (1 of note:5000) it, is incubated at room temperature 30min, after TBST washes film 3 times plus result is observed in DAB colour developing.As a result It is as Figure 1-Figure 5 respectively.As shown, be 0.33mmol/L with IPTG concentration, after inducing expression 4-5h, expressing quantity It is higher.
The purifying of 5.VP1 albumen and immunogenic polypeptide
The expression thallus of IPTG induction is collected by centrifugation, thallus (every 50mL thallus adds 3mLPBS) is resuspended in PBS, according to 1:100 Ultrasonication in 100mmol/L phenylmethylsulfonyl fluoride (PMSF) ice bath is added in ratio, and 10000r/min centrifugation abandons supernatant, will sink Shallow lake is resuspended in 19.7mL Buffer A (Tris-base：50mM,EDTA：0.5mM,NaCl：50mM, glycerol：5%, DTT 5mM) and the sarcosyl of 0.3mL 20% (SKL) stores liquid, and vigorous agitation is slowly dissolved it, is stored at room temperature To solubilization of inclusion bodies；4 DEG C of 10000r/min are centrifuged 10 minutes, take supernatant；Add 20% polyethylene glycol (PEG4000) 210ul and The oxidized form of glutathione 420ul of 50mM adds the reduced glutathione 420ul of 100mM, 30min to 2h is stood, with PBS (pH8.0) it dialyses, then with anti-GST affinity chromatography column purification, operating process strictly observes specification.Determination of protein concentration uses BCA protein quantification kit.
Embodiment 2：The antigenicity identification of VP1 albumen and immunogenic polypeptide
1.Western Blot method identification antigenicity：
Protein sample prepared by embodiment 1 is by 1:4 are added 5 × SDS-PAGE sample-loading buffer, boiling water bath after mixing 7min, 4 DEG C of 10000r/min are centrifuged 2min, after taking supernatant to carry out electrophoresis, by bacterium solution protein delivery to nitrocellulose filter, 5% skimmed milk power closes 40min；The anti-EV71 of primary antibody source of mouse mostly anti-(1:5000) 4 DEG C of overnight incubations, after TBST washes film 3 times, The goat anti-mouse IgG antibodies (1 of secondary antibody addition horseradish peroxidase-labeled:5000) it, is incubated at room temperature 30min, TBST washes film 3 After secondary plus result is observed in DAB colour developing.Simultaneously using pET-49b (+) empty carrier convert to DE3 Escherichia coli inducing expression product as Negative control.As a result respectively as shown in Fig. 6-Figure 10.As shown, protein purification effect is preferable, identified, the albumen of purifying is equal With antigenicity.
2.ELISA method identification antigenicity：
Protein sample prepared by embodiment 1 is coated on ELISA coating plate, every kind of albumen sets up two dilutions, makes (coating buffer is diluted with coating buffer：Contain Na in every 1L distilled water2CO3:1.59g,NaHCO3:2.93g), first A dilution protein concentration is：2.08×10-2Mg/mL, second dilution protein concentration are：1.04×10-2mg/mL；Albumen It is stayed overnight for 4 DEG C after coating；Next day PBST board-washing 3 times, each 3min；5% bovine serum albumin(BSA) (BSA), 37 DEG C of closing 1h；PBST Board-washing 3 times, each 3min；The anti-EV71 of primary antibody source of mouse mostly anti-(1：5000) 37 DEG C of incubation 2h；PBST board-washing 6 times, every time 3min；The goat anti-mouse IgG antibodies (1 of secondary antibody addition horseradish peroxidase-labeled：5000), 37 DEG C of incubation 1h；PBST board-washing 6 times, each 3min；The colour developing of TMB one-component developing solution.PET-49b (+) empty carrier is converted to DE3 Escherichia coli simultaneously and is induced Expression product is as negative control.Testing result is as shown in table 2.
2 ELISA method of table identifies detection of antigenicity result
As shown in table 2, using GST albumen as negative control, no matter dilution 1 or dilution 2, VP1 antigen and its immunogene Property polypeptide A value have 2 times or more growth compared with the A value of GST albumen, be accredited as positive findings, i.e., purified albumen has antigen Property.
Embodiment 3：The identification of the immunogenicity of VP1 albumen and immunogenic polypeptide
By the VP1 albumen after purification of embodiment 1 and immunogenic polypeptide and Freund's adjuvant according to volume ratio 1:1 ratio mixes Balb/c mouse is immunized afterwards, 5 mouse of every kind of protein immunization are immunized 4 times altogether.Initial immunity 1.0mg albumen Jia Fushi is helped completely Agent is immunized 0.5mg albumen and adds incomplete Freund's adjuvant, is spaced 1 week continuous immunity 0.5mg albumen later and adds Freund endless after 2 weeks Full adjuvant twice, plucks eyeball in final immunization after a week and takes blood；It is small using pET-49b (+) empty carrier expression protein immunization simultaneously Mouse takes blood as negative control.Whether Western Blot detection serum and EV71 virus can occur specific reaction, with inspection Survey protein immunogenic.As a result as shown in figure 11.
As shown, the EV71 virus of RD cell culture is added in the loading hole of polyacrylamide gel, by RD cell Centrifugation gained supernatant, which is added in loading hole, makees negative control, with institute after the immune BALB/c mouse of the VP1 albumen and its polypeptide of purifying Obtaining serum is primary antibody, EV71 virus, VP1-GST fusion protein, VP179-432Gst fusion protein, VP1436-864GST merges egg White, VP1436-735Gst fusion protein and VP1565-864Gained serum can be sent out with EV71 virus after mouse is immunized in gst fusion protein Raw specific binding, band molecular weight as shown in the figure is about 35KD (EV71 Structural protein VP1)；Obtained by GST protein immunization mouse Serum can also be specifically bound with EV71 virus, but the non-EV71 Structural protein VP1 of band molecular size range as shown in the figure； Serum obtained by mouse and EV71 virus is immunized without specific binding (negative control) in PBS.
SEQUENCE LISTING
<110>Shandong University
Shandong Province medical device product quality inspection center
<120>The immunogenic polypeptide and the preparation method and application thereof of the VP1 antigen of enterovirns type 71
<130> 2015
<160> 20
<170> PatentIn version 3.5
<210> 1
<211> 297
<212> PRT
<213>SDLY107 plants of VP1 amino acid sequences
<400> 1
Gly Asp Arg Val Ala Asp Val Ile Glu Ser Ser Ile Gly Asp Ser Val
1 5 10 15
Ser Arg Ala Leu Thr His Ala Leu Pro Ala Pro Thr Gly Gln Asn Thr
20 25 30
Gln Val Ser Ser His Arg Leu Asp Thr Gly Lys Val Pro Ala Leu Gln
35 40 45
Ala Ala Glu Ile Gly Ala Ser Ser Asn Ala Ser Asp Glu Ser Met Ile
50 55 60
Glu Thr Arg Cys Val Leu Asn Ser His Ser Thr Ala Glu Thr Thr Leu
65 70 75 80
Asp Ser Phe Phe Ser Arg Ala Gly Leu Val Gly Glu Ile Asp Leu Pro
85 90 95
Leu Lys Gly Thr Thr Asn Pro Asn Gly Tyr Ala Asn Trp Asp Ile Asp
100 105 110
Ile Thr Gly Tyr Ala Gln Met Arg Arg Lys Val Glu Leu Phe Thr Tyr
115 120 125
Met Arg Phe Asp Ala Glu Phe Thr Phe Val Ala Cys Thr Pro Thr Gly
130 135 140
Glu Val Val Pro Gln Leu Leu Gln Tyr Met Phe Val Pro Pro Gly Ala
145 150 155 160
Pro Lys Pro Asp Ser Arg Glu Ser Leu Ala Trp Gln Thr Ala Thr Asn
165 170 175
Pro Ser Val Phe Val Lys Leu Ser Asp Pro Pro Ala Gln Val Ser Val
180 185 190
Pro Phe Met Ser Pro Ala Ser Ala Tyr Gln Trp Phe Tyr Asp Gly Tyr
195 200 205
Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu Glu Tyr Gly Ala
210 215 220
Cys Pro Asn Asn Met Met Gly Thr Phe Ser Val Arg Thr Val Gly Thr
225 230 235 240
Ser Lys Ser Lys Tyr Pro Leu Val Val Arg Ile Tyr Met Arg Met Lys
245 250 255
His Val Arg Ala Trp Ile Pro Arg Pro Met Arg Asn Gln Asn Tyr Leu
260 265 270
Phe Lys Ala Asn Pro Asn Tyr Ala Gly Asn Ser Ile Lys Pro Thr Gly
275 280 285
Ala Ser Arg Thr Ala Ile Thr Thr Leu
290 295
<210> 2
<211> 118
<212> PRT
<213>The immunogenic polypeptide of VP1 antigen（Amino acid sequence corresponding to gene 79-432）
<400> 2
Pro Thr Gly Gln Asn Thr Gln Val Ser Ser His Arg Leu Asp Thr Gly
1 5 10 15
Lys Val Pro Ala Leu Gln Ala Ala Glu Ile Gly Ala Ser Ser Asn Ala
20 25 30
Ser Asp Glu Ser Met Ile Glu Thr Arg Cys Val Leu Asn Ser His Ser
35 40 45
Thr Ala Glu Thr Thr Leu Asp Ser Phe Phe Ser Arg Ala Gly Leu Val
50 55 60
Gly Glu Ile Asp Leu Pro Leu Lys Gly Thr Thr Asn Pro Asn Gly Tyr
65 70 75 80
Ala Asn Trp Asp Ile Asp Ile Thr Gly Tyr Ala Gln Met Arg Arg Lys
85 90 95
Val Glu Leu Phe Thr Tyr Met Arg Phe Asp Ala Glu Phe Thr Phe Val
100 105 110
Ala Cys Thr Pro Thr Gly
115
<210> 3
<211> 143
<212> PRT
<213>The immunogenic polypeptide of VP1 antigen（Amino acid sequence corresponding to gene 436-864）
<400> 3
Val Val Pro Gln Leu Leu Gln Tyr Met Phe Val Pro Pro Gly Ala Pro
1 5 10 15
Lys Pro Asp Ser Arg Glu Ser Leu Ala Trp Gln Thr Ala Thr Asn Pro
20 25 30
Ser Val Phe Val Lys Leu Ser Asp Pro Pro Ala Gln Val Ser Val Pro
35 40 45
Phe Met Ser Pro Ala Ser Ala Tyr Gln Trp Phe Tyr Asp Gly Tyr Pro
50 55 60
Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu Glu Tyr Gly Ala Cys
65 70 75 80
Pro Asn Asn Met Met Gly Thr Phe Ser Val Arg Thr Val Gly Thr Ser
85 90 95
Lys Ser Lys Tyr Pro Leu Val Val Arg Ile Tyr Met Arg Met Lys His
100 105 110
Val Arg Ala Trp Ile Pro Arg Pro Met Arg Asn Gln Asn Tyr Leu Phe
115 120 125
Lys Ala Asn Pro Asn Tyr Ala Gly Asn Ser Ile Lys Pro Thr Gly
130 135 140
<210> 4
<211> 100
<212> PRT
<213>The immunogenic polypeptide of VP1 antigen（Amino acid sequence corresponding to gene 436-735）
<400> 4
Val Val Pro Gln Leu Leu Gln Tyr Met Phe Val Pro Pro Gly Ala Pro
1 5 10 15
Lys Pro Asp Ser Arg Glu Ser Leu Ala Trp Gln Thr Ala Thr Asn Pro
20 25 30
Ser Val Phe Val Lys Leu Ser Asp Pro Pro Ala Gln Val Ser Val Pro
35 40 45
Phe Met Ser Pro Ala Ser Ala Tyr Gln Trp Phe Tyr Asp Gly Tyr Pro
50 55 60
Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu Glu Tyr Gly Ala Cys
65 70 75 80
Pro Asn Asn Met Met Gly Thr Phe Ser Val Arg Thr Val Gly Thr Ser
85 90 95
Lys Ser Lys Tyr
100
<210> 5
<211> 100
<212> PRT
<213>The immunogenic polypeptide of VP1 antigen（Amino acid sequence corresponding to gene 565-864）
<400> 5
Gln Val Ser Val Pro Phe Met Ser Pro Ala Ser Ala Tyr Gln Trp Phe
1 5 10 15
Tyr Asp Gly Tyr Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu
20 25 30
Glu Tyr Gly Ala Cys Pro Asn Asn Met Met Gly Thr Phe Ser Val Arg
35 40 45
Thr Val Gly Thr Ser Lys Ser Lys Tyr Pro Leu Val Val Arg Ile Tyr
50 55 60
Met Arg Met Lys His Val Arg Ala Trp Ile Pro Arg Pro Met Arg Asn
65 70 75 80
Gln Asn Tyr Leu Phe Lys Ala Asn Pro Asn Tyr Ala Gly Asn Ser Ile
85 90 95
Lys Pro Thr Gly
100
<210> 6
<211> 891
<212> DNA
<213>VP1 full-length gene
<400> 6
ggagataggg tggcagatgt aattgaaagt tccataggag atagcgtgag cagagccctc 60
actcacgctc taccagcacc cacaggccag aacacacagg tgagcagtca tcgactggat 120
acaggcaagg ttccagcact ccaagctgct gaaattggag catcatcaaa tgctagtgac 180
gagagcatga ttgagacacg ctgtgtcctt aactcgcaca gtacagctga gaccactctt 240
gatagtttct tcagtagggc gggattagtt ggagagatag atctccctct taagggcaca 300
actaacccaa atggttatgc caactgggac atagatataa caggttacgc gcaaatgcgt 360
agaaaggtag agctattcac ctacatgcgc tttgatgcag agttcacttt tgttgcgtgc 420
acacccaccg gggaagttgt cccacaattg ctccaatata tgtttgtgcc acccggagcc 480
cctaagccag attctaggga atcccttgca tggcaaaccg ccactaaccc ctcagttttt 540
gtcaagctgt cagaccctcc tgcgcaggtt tcagtgccat tcatgtcacc tgcgagtgct 600
tatcaatggt tttatgacgg atatcccaca ttcggagaac acaaacagga gaaagatctt 660
gaatacgggg catgtcctaa taacatgatg ggcacgttct cagtgcggac tgtggggacc 720
tccaagtcca agtacccttt agtggttagg atttacatga gaatgaagca cgtcagggcg 780
tggatacctc gcccgatgcg taaccagaac tacctattca aagccaaccc aaattatgct 840
ggcaactcca ttaagccaac tggtgccagt cgcacagcga tcaccactct t 891
<210> 7
<211> 354
<212> DNA
<213> VP1（79-432）Gene
<400> 7
cccacaggcc agaacacaca ggtgagcagt catcgactgg atacaggcaa ggttccagca 60
ctccaagctg ctgaaattgg agcatcatca aatgctagtg acgagagcat gattgagaca 120
cgctgtgtcc ttaactcgca cagtacagct gagaccactc ttgatagttt cttcagtagg 180
gcgggattag ttggagagat agatctccct cttaagggca caactaaccc aaatggttat 240
gccaactggg acatagatat aacaggttac gcgcaaatgc gtagaaaggt agagctattc 300
acctacatgc gctttgatgc agagttcact tttgttgcgt gcacacccac cggg 354
<210> 8
<211> 429
<212> DNA
<213> VP1（436-864）Gene
<400> 8
gttgtcccac aattgctcca atatatgttt gtgccacccg gagcccctaa gccagattct 60
agggaatccc ttgcatggca aaccgccact aacccctcag tttttgtcaa gctgtcagac 120
cctcctgcgc aggtttcagt gccattcatg tcacctgcga gtgcttatca atggttttat 180
gacggatatc ccacattcgg agaacacaaa caggagaaag atcttgaata cggggcatgt 240
cctaataaca tgatgggcac gttctcagtg cggactgtgg ggacctccaa gtccaagtac 300
cctttagtgg ttaggattta catgagaatg aagcacgtca gggcgtggat acctcgcccg 360
atgcgtaacc agaactacct attcaaagcc aacccaaatt atgctggcaa ctccattaag 420
ccaactggt 429
<210> 9
<211> 300
<212> DNA
<213> VP1（436-735）Gene
<400> 9
gttgtcccac aattgctcca atatatgttt gtgccacccg gagcccctaa gccagattct 60
agggaatccc ttgcatggca aaccgccact aacccctcag tttttgtcaa gctgtcagac 120
cctcctgcgc aggtttcagt gccattcatg tcacctgcga gtgcttatca atggttttat 180
gacggatatc ccacattcgg agaacacaaa caggagaaag atcttgaata cggggcatgt 240
cctaataaca tgatgggcac gttctcagtg cggactgtgg ggacctccaa gtccaagtac 300
<210> 10
<211> 300
<212> DNA
<213> VP1（565-864）Gene
<400> 10
caggtttcag tgccattcat gtcacctgcg agtgcttatc aatggtttta tgacggatat 60
cccacattcg gagaacacaa acaggagaaa gatcttgaat acggggcatg tcctaataac 120
atgatgggca cgttctcagt gcggactgtg gggacctcca agtccaagta ccctttagtg 180
gttaggattt acatgagaat gaagcacgtc agggcgtgga tacctcgccc gatgcgtaac 240
cagaactacc tattcaaagc caacccaaat tatgctggca actccattaa gccaactggt 300
<210> 11
<211> 34
<212> DNA
<213>Artificial sequence
<400> 11
cggactagtg gagatagggt ggcagatgta attg 34
<210> 12
<211> 33
<212> DNA
<213>Artificial sequence
<400> 12
cccaagcttc taaagagtgg tgatcgctgt gcg 33
<210> 13
<211> 37
<212> DNA
<213>Artificial sequence
<400> 13
cggactagta tgcccacagg ccagaacaca caggtga 37
<210> 14
<211> 37
<212> DNA
<213>Artificial sequence
<400> 14
cccaagcttc tacccggtgg gtgtgcacgc aacaaaa 37
<210> 15
<211> 37
<212> DNA
<213>Artificial sequence
<400> 15
cggactagta tggttgtccc acaattgctc caatata 37
<210> 16
<211> 34
<212> DNA
<213>Artificial sequence
<400> 16
cccaagcttc taaccagttg gcttaatgga gttg 34
<210> 17
<211> 32
<212> DNA
<213>Artificial sequence
<400> 17
cggactagtg ttgtcccaca attgctccaa ta 32
<210> 18
<211> 32
<212> DNA
<213>Artificial sequence
<400> 18
cccaagcttc tagtacttgg acttggaggt cc 32
<210> 19
<211> 32
<212> DNA
<213>Artificial sequence
<400> 19
cggactagtc aggtttcagt gccattcatg tc 32
<210> 20
<211> 33
<212> DNA
<213>Artificial sequence
<400> 20
cccaagcttc taaccagttg gcttaatgga gtt 33

Claims (9)

1. a kind of immunogenic polypeptide of enterovirns type 71 VP1 antigen, is characterized in that, amino acid sequence such as SEQ ID Shown in NO.4.
2. encoding the polynucleotides of immunogenic polypeptide described in claim 1, which is characterized in that the sequence of the polynucleotides As shown in SEQ ID NO.9.
3. a kind of recombinant vector contains polynucleotides as claimed in claim 2.
4. recombinant vector as claimed in claim 3, is characterized in that, the carrier that sets out is pET-49b (+).
5. a kind of recombinant expression bacterium, contains recombinant vector as claimed in claim 4.
6. recombinant expression bacterium as claimed in claim, is characterized in that, starting strain is Escherichia coli DE3.
7. a kind of preparation method for preparing immunogenic polypeptide described in claim 1, which is characterized in that include the following steps：
(1) by genetic fragment shown in SEQ ID NO.9 and carrier pET-49b (+), after III double digestion of SpeI and Hand, even It connects building and obtains expression vector pET-49b (+)-VP1436-735；
(2) by expression vector pET-49b (+)-VP1 of step (1)436-735To Escherichia coli DE3, screening positive clone is lured for conversion Lead expression recombinant protein；
(3) by the recombinant protein of step (2) using glutathione sulfydryl transferase agarose gel purification to get.
8. preparation method as claimed in claim 7, which is characterized in that in step (1), gene piece shown in SEQ ID NO.9 Section construction method be：Using SDLY107 full-length cDNA as pcr template, to draw shown in SEQ ID NO.17-SEQ ID NO.18 Object sequence is amplimer, is obtained through PCR amplification.
9. application of the immunogenic polypeptide described in claim 1 in the diagnostic reagent for preparing EV71 virus.
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