CN108912213B - Immunogenic polypeptide of enterovirus 71 type VP1 antigen and preparation method and application thereof - Google Patents
Immunogenic polypeptide of enterovirus 71 type VP1 antigen and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of immunobiology, and relates to an immunogenic polypeptide of an enterovirus 71 type VP1 antigen, and a preparation method and application thereof. In particular to an immunogenic polypeptide of an enterovirus 71 type VP1 antigen, which has an amino acid sequence shown as any sequence of SEQ ID NO.1-SEQ ID NO.5 in a sequence table, or an amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence. The invention also relates to a preparation method of the immunogenic polypeptide and application of the immunogenic polypeptide in preparation of a diagnostic reagent of EV71 virus. The immunogenic polypeptide has good antigenicity and immunogenicity, and provides a research basis for early diagnosis reagent development and vaccine development of EV 71.
Description
Technical Field
The invention belongs to the technical field of immunobiology, and particularly relates to an immunogenic polypeptide of an enterovirus 71 type VP1 antigen, and a preparation method and application thereof.
Background
EV71 is a single-stranded positive-strand RNA virus belonging to Enterovirus A genus of picornaviridae, and its infection can cause hand-foot-and-mouth disease of infants, and the pathogenesis is mainly manifested by fever, diarrhea and herpangina. Unlike typical hand-foot-and-mouth disease, infection with certain strain EV71 can cause severe hand-foot-and-mouth disease with complications including aseptic meningitis, encephalitis, polio-like paralysis, neurogenic pulmonary edema, and even death. The EV71 has wide infection, the prevalence range of the EV71 is wide, the death rate of the EV71 is high, and the EV71 is a public health problem which is valued at present.
Over the years, various national scholars have been dedicated to the development of EV71 vaccines, and with the cross development of multiple subjects such as molecular biology, immunology and the like, recombinant subunit vaccines, DNA vaccines and virus-like particle vaccines of EV71 are developed successively. The conventional EV71 full-killed vaccine is developed most rapidly, and three organizations in China have completed phase III clinical tests of the EV71 full-killed vaccine at present. However, the whole inactivated vaccine has many problems, such as generally low immune effect, incapability of inducing cytotoxic T lymphocyte reaction, short duration of immune reaction generated by induction, influence of an inactivating agent on a virus antigen, and the like; there is therefore a great need to develop new vaccines that effectively stimulate the immune response while retaining intact immunogenicity, while avoiding infection.
Regarding the infection diagnosis of EV71, the traditional method comprises a neutralization experiment method and a virus separation method, and the methods are time-consuming and labor-consuming, have high experiment requirements and are not suitable for clinical application. In recent years, molecular biology detection technologies (such as reverse transcription polymerase chain reaction (RT-PCR) and Real-time fluorescence Quantitative polymerase chain reaction (Quantitative Real-time PCR)) are increasingly applied to diagnosis of EV71 infection, but such methods have false positive and false negative, have high requirements for experimental technologies, and are not easy to develop in primary hospitals. Enzyme-linked immunosorbent assay (ELISA) and Immunochromatography (ICA) utilize the principle of antigen-antibody combination to detect EV71, the operation is simple and rapid, and at present, many related researches are carried out, but the detection is not popularized clinically.
EV71 can be divided into A, B, C subtypes, wherein subtypes B and C can be respectively divided into B1-B5 and C1-C5. EV71 contains only one Open Reading Frame (ORF), 5 'UTR and 3' UTR upstream and downstream of the ORF, respectively. The total length of the genome RNA contains 7500 nucleotides, encodes polyprotein with 2193 amino acids, and the polyprotein can be hydrolyzed into three precursor proteins of P1, P2 and P3 and finally into 4 structural proteins (VP1-VP4) and 7 non-structural proteins (2A-2C and 3A-3D). Of the 4 structural proteins, VP4 is embedded inside the viral coat, and the remaining three are exposed on the surface of the viral particle and directly contact the host immune system; the VP1 protein has genetic diversity corresponding to serotype and contains main antigenic determinant of EV71, so VP1 is one of the hot spots of the current research and has been widely applied to the pathogen detection and vaccine research of hand-foot-and-mouth disease.
The BC loop region (amino acid positions 91-106) of the VP1 protein is a currently accepted neutralizing epitope site, but whether other important epitopes exist or not needs further research and demonstration.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide an immunogenic polypeptide of an enterovirus 71 type VP1 antigen, and a preparation method and application thereof. The VP1 protein is segmented, the antigenicity and immunogenicity of each peptide segment are researched, and a basic research is provided for the development of early diagnosis reagents and vaccines of EV 71.
In order to achieve the purpose, the invention adopts the following technical scheme:
one aspect of the invention relates to an immunogenic polypeptide of an enterovirus 71 type VP1 antigen, which has an amino acid sequence shown as any one of SEQ ID NO.1-SEQ ID NO.5 in a sequence table, or an amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence.
Yet another aspect of the invention relates to polynucleotides encoding the above immunogenic polypeptides; specifically, the sequences of the polynucleotides are respectively shown as SEQ ID NO.6-SEQ ID NO. 10.
The invention also provides a recombinant vector containing the polynucleotide sequence.
The starting vector of the recombinant vector is pET-49b (+).
The invention also provides a recombinant expression bacterium containing the recombinant vector.
The starting strain of the recombinant expression strain is Escherichia coli DE 3.
The invention also provides a method for expressing and purifying immunogenic polypeptides of the enterovirus 71 type VP1 antigen, which comprises the following steps:
(1) the gene fragment shown in SEQ ID NO.6-SEQ ID NO.10 and the vector pET-49b (+) are subjected to SpeI and Hand III double enzyme digestion and are connected to construct expression vectors pET-49b (+) -VP1 and pET-49b (+) -VP179-432、pET-49b(+)-VP1436-864、pET-49b(+)-VP1436-735And pET-49b (+) -VP1565-864；
(2) The expression vectors pET-49b (+) -VP1, pET-49b (+) -VP1 of the step (1)79-432、pET-49b(+)-VP1436-864、pET-49b(+)-VP1436-735And pET-49b (+) -VP1565-864Transforming to Escherichia coli DE3, screening positive clone, and inducing to express recombinant protein;
(3) and (3) purifying the recombinant protein in the step (2) by adopting glutathione mercaptotransferase (GST) agarose gel to obtain the glutathione mercaptotransferase.
In the step (1), the construction method of the gene segments shown by SEQ ID NO.6-SEQ ID NO.10 is as follows: taking SDLY107(GenBank: JX244186.1) full-length cDNA as a PCR template, taking a primer sequence shown in SEQ ID NO.11-SEQ ID NO.20 as an amplification primer, and carrying out PCR amplification to obtain a VP1 gene full-length fragment and 4 segments of VP1 gene fragments which are respectively VP1 and VP179-432、VP1436-864、VP1436-735And VP1565-864。
The amplified primer sequences are specifically 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 invention also provides application of the immunogenic polypeptide of the VP1 antigen of the enterovirus 71 in preparation of a diagnostic reagent of EV71 virus.
The invention has the beneficial effects that:
(1) the invention discovers a novel immunogenic polypeptide outside a BC loop region (91-106 amino acid sites) on EV71 structural protein VP1, and the polypeptide has good antigenicity and immunogenicity, thereby providing a research basis for early diagnosis reagent development and vaccine development of EV 71.
(2) The expression and purification method of the immunogenic polypeptide is simple, convenient and time-saving to operate, and is beneficial to realizing large-scale industrial production.
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FIG. 1: expression and identification results of VP1 protein; in the figure, 1: protein marker; 2: DE3 cells which have not been transformed with a plasmid; 3: inducing and expressing for 3h by IPTG with the concentration of 0.33 mmol/L; 4: inducing and expressing for 3h by IPTG with the concentration of 0.67 mmol/L; 5: inducing and expressing for 4h by IPTG with the concentration of 0.33 mmol/L; 6: inducing and expressing for 4h by IPTG with the concentration of 0.67 mmol/L; 7: inducing and expressing for 5h by IPTG with the concentration of 0.33 mmol/L; 8: the expression is induced by IPTG with the concentration of 0.67mmol/L for 5 h. The molecular weight of the VP1-GST fusion protein is 59 kd.
FIG. 2: immunogenic polypeptide VP179-432Expression and identification results of (a); in the figure, 1: protein marker; 2: inducing and expressing for 3h by IPTG with the concentration of 0.33 mmol/L; 3: inducing and expressing for 3h by IPTG with the concentration of 0.67 mmol/L; 4: inducing and expressing for 4h by IPTG with the concentration of 0.33 mmol/L; 5: inducing and expressing for 4h by IPTG with the concentration of 0.67 mmol/L; 6: inducing and expressing for 5h by IPTG with the concentration of 0.33 mmol/L; 7: the expression is induced by IPTG with the concentration of 0.67mmol/L for 5 h. VP179-432The molecular weight of the GST fusion protein is about 39 kd; 8: DE3 cells which were not transformed with the plasmid.
FIG. 3: immunogenic polypeptide VP1436-864Expression and identification results of (a); in the figure, 1: protein marker; 2: inducing and expressing for 3h by IPTG with the concentration of 0.33 mmol/L; 3: inducing and expressing for 3h by IPTG with the concentration of 0.67 mmol/L; 4: inducing and expressing for 4h by IPTG with the concentration of 0.33 mmol/L; 5: inducing and expressing for 4h by IPTG with the concentration of 0.67 mmol/L; 6: inducing and expressing for 5h by IPTG with the concentration of 0.33 mmol/L; 7: the IPTG induction expression with the concentration of 0.67mmol/L5h。VP1436-864The molecular weight of the GST fusion protein is about 40 kd; 8: DE3 cells which were not transformed with the plasmid.
FIG. 4: immunogenic polypeptide VP1436-735Expression and identification results of (a); in the figure, 1: protein marker; 2: DE3 cells which have not been transformed with a plasmid; 3: inducing and expressing for 3h by IPTG with the concentration of 0.33 mmol/L; 4: inducing and expressing for 3h by IPTG with the concentration of 0.67 mmol/L; 5: inducing and expressing for 4h by IPTG with the concentration of 0.33 mmol/L; 6: inducing and expressing for 4h by IPTG with the concentration of 0.67 mmol/L; 7: inducing and expressing for 5h by IPTG with the concentration of 0.33 mmol/L; 8: the expression is induced by IPTG with the concentration of 0.67mmol/L for 5 h. VP1436-735The molecular weight of the GST fusion protein is 40 kd.
FIG. 5: immunogenic polypeptide VP1565-864Expression and identification results of (a); in the figure, 1: protein marker; 2: DE3 cells which have not been transformed with a plasmid; 3: inducing and expressing for 3h by IPTG with the concentration of 0.33 mmol/L; 4: inducing and expressing for 3h by IPTG with the concentration of 0.67 mmol/L; 5: inducing and expressing for 4h by IPTG with the concentration of 0.33 mmol/L; 6: inducing and expressing for 4h by IPTG with the concentration of 0.67 mmol/L; 7: inducing and expressing for 5h by IPTG with the concentration of 0.33 mmol/L; 8: the expression is induced by IPTG with the concentration of 0.67mmol/L for 5 h. VP1565-864The molecular weight of the GST fusion protein is 37 kd.
FIG. 6: the result of the antigenicity detection of the purified VP1 protein; in the figure, 1: negative control; 2: the molecular weight of the purified VP1-GST fusion protein, VP1-GST fusion protein is 59 kd; 3: unpurified VP1-GST fusion protein; the primary antibody was murine anti-EV 71 serum.
FIG. 7: purified VP179-432Detecting the antigenicity of the protein; in the figure, 1: unpurified VP179-432GST fusion protein, VP179-432The molecular weight of the GST fusion protein is about 39 kd; 2-3: purified VP179-432-a GST fusion protein; 4: negative control; the primary antibody was murine anti-EV 71 serum.
FIG. 8: purified VP1436-864Detecting the antigenicity of the protein; in the figure, 1: unpurified VP1436-864GST fusion protein, VP1436-864The molecular weight of the GST fusion protein is about 40 kd; 2: purified VP1436-864-a GST fusion protein; 3: negative control(ii) a Primary antibody was murine anti-EV 71 serum.
FIG. 9: purified VP1436-735Detecting the antigenicity of the protein; in the figure, 1: unpurified VP1436-735GST fusion protein, VP1436-735The molecular weight of the GST fusion protein is about 40 kd; 2: purified VP1436-735-a GST protein; 3: negative control; primary antibody was murine anti-EV 71 serum.
FIG. 10: purified VP1565-864Detecting the antigenicity of the protein; in the figure, 1: unpurified VP1565-864GST fusion protein, VP1565-864The molecular weight of the GST fusion protein is about 37 kd; 2: purified VP1565-864-a GST protein; 3: negative control; primary antibody was murine anti-EV 71 serum.
FIG. 11: the identification result of the immunogenicity of the VP1 protein and the immunogenic polypeptide; in the figure, 1, 2 and 3 are EV71 virus, negative control and protein marker, respectively. Negative control is the supernatant after centrifugation of the RD cells.
Detailed Description
The present invention will be further described with reference to examples, but the following description is only for the purpose of explaining the present invention and does not limit the contents thereof.
The experimental materials and reagents used in the following examples are as follows:
1. experimental materials:
1.1 cells, viruses and laboratory animals
The cells used were human rhabdomyosarcoma cells (RD). Inoculating EV71SDLY107 virus when RD cells grow to a logarithmic phase, repeatedly freezing and thawing for three times at-40 ℃ and room temperature when cytopathic rate reaches about 80%, centrifuging for 10 minutes at 4 ℃ and 10,000r/min, collecting the supernatant, harvesting the virus, and storing the virus in a refrigerator at-80 ℃. The total number of Balb/c mice is 25, and the number of Kunming mice is 5, and the Balb/c mice are purchased from the experimental animal center of Shandong university.
1.2 Experimental reagents
The main reagents used are viral RNA extraction kit (OMEGA, China), reverse transcription kit (TOYOBO, Japan), DNA gel recovery kit and plasmid extraction kit (OMEGA, China); cell culture medium, serum (HyClone, usa); LA Taq enzyme, T4DNA ligase, DNA Marker (Takara, china); restriction enzymes, protein markers (Thermo, usa); PVDF membranes (Millipore, usa); 5 × protein loading buffer (bi yun day, china); BCA protein quantification kit (edley, china); horseradish enzyme labeled goat anti-mouse IgG, concentrated DAB kit (China ); GST monoclonal antibody (Proteintech, usa); TMB single-component color developing solution (solibao, china); GST sepharose (well in the century, china); the primer is synthesized by Shanghai biological engineering Co., Ltd; sequencing was performed by Shanghai Boshang Biotech.
Example 1: expression and purification of immunogenic polypeptides of VP1 antigen of enterovirus type 71
1. Amplification of cDNA of EV71SDLY107 strain virus
RNA of SDLY107 strain virus was extracted using the Viral RNA Kit from OMEGA, SDLY107 strain was isolated from children with hand-foot-and-mouth disease who died in Min Hospital, Linyi, Shandong province, and was identified as a virulent strain (after the strain infected cells, the cell disease became fast and strong, and the pathological characteristics of animals were evident), which had been subjected to full sequence analysis (GenBank: JX244186.1), and cDNA was generated by reverse transcription using the ReverTra Ace qPCR Kit from TOYOBO.
2. Primer design and PCR amplification
Using SDLY107 full-length cDNA as a PCR template, designing primers for PCR amplification, wherein the sequences of the primers are shown in Table 1:
TABLE 1 primer sequences
Amplifying a VP1 full-length gene (shown as SEQ ID NO. 6) of an SDLY107 strain by using the SDLY107 full-length cDNA as a PCR template, using SpeI VP1 Primer F as an upstream Primer and using HindIII VP1 Primer R as a downstream Primer; 4 segments of VP1 gene fragments are amplified by using 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 as primers, namely: VP179-432(shown as SEQ ID NO. 7), VP1436-864(shown as SEQ ID NO. 8), VP1436-735(shown as SEQ ID NO. 9), VP1565-864(shown as SEQ ID NO. 10).
PCR conditions were as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 30 s; annealing at 55 ℃ for 30 s; extending for 1min at 72 ℃; final extension at 72 ℃ for 10min for 30 cycles.
3. Construction of expression vectors
SpeI and HindIII double digestion of pET-49b (+) vector and "2. primer design and PCR amplification" DNA fragment obtained: 2. mu.L of each of SpeI and HindIII Fastdigest enzyme, 10. mu.g of DNA fragment, 5. mu.L of 10 XFastdigest Green Buffer, and 50. mu.L of water were replenished, and digested at 37 ℃ for 4 hours. Recovering the cut fragments from the gel, and connecting the fragments overnight by using T4 ligase to obtain recombinant plasmids pET-49b (+) -VP1 and pET-49b (+) -VP179-432、pET-49b(+)-VP1436-864、pET-49b(+)-VP1436-735、pET-49b(+)-VP1565-864。
Expression and identification of VP1 protein and immunogenic polypeptide
10. mu.L of the recombinant plasmid constructed above was transformed into 100. mu.L of E.coli DE3, added into 890. mu.L of LB liquid medium at 37 ℃ for 1 hour at 160rpm, 100. mu.L of the bacterial solution was spread evenly on solid agar medium and left overnight at 37 ℃. Selecting positive colonies screened by kanamycin (30 mu g/mL) the next day, inoculating the positive colonies into 5mL LB (containing kanamycin) liquid culture medium, and performing shake culture at 37 ℃ and 160rpm overnight; inoculating the strain into a fresh LB liquid culture medium according to the proportion of 1:100 the next day, carrying out shake culture at 37 ℃ until the OD value reaches 0.8-1.0, adding 0.33mmol/L isopropyl-beta-D-thiogalactopyranoside (IPTG), continuing to culture for 4-5h, taking 1mL bacterial liquid, centrifuging at 4 ℃ 10000r/min for 2min, collecting thalli, adding 80 mu L Phosphate Buffer Solution (PBS) to resuspend the thalli, and mixing the samples according to the proportion of 1:4 adding 5 xSDS-PAGE sample buffer solution, mixing uniformly, carrying out boiling water bath for 7min, centrifuging at 4 ℃ at 10000r/min for 2min, taking supernatant, carrying out electrophoresis, transferring bacterial liquid protein onto a nitrocellulose membrane, and sealing with 5% skimmed milk powder for 40 min; incubating the primary antibody with rabbit-derived anti-GST antibody (1:5000) at 4 ℃ overnight, washing the membrane with TBST for 3 times, adding horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:5000) into the secondary antibody, incubating at room temperature for 30min, washing the membrane with TBST for 3 times, and adding DAB for color observation. The results are shown in FIGS. 1 to 5, respectively. As shown in the figure, the protein expression amount is higher after induction expression is carried out for 4-5h by using the IPTG concentration of 0.33 mmol/L.
Purification of VP1 protein and immunogenic polypeptides
Centrifugally collecting IPTG induced expression thallus, re-suspending the thallus with PBS (3 mL of PBS is added to each 50mL of thallus), adding 100mmol/L of phenylmethylsulfonyl fluoride (PMSF) according to the proportion of 1:100, ultrasonically crushing in ice bath, centrifuging at 10000r/min, discarding supernatant, re-suspending the precipitate in 19.7mL of Buffer A (Tris-base: 50mM, EDTA: 0.5mM, NaCl: 50mM, glycerol: 5%, DTT 5mM) and 0.3mL of 20% sodium lauryl Sarcosinate (SKL) stock solution, violently stirring to slowly dissolve the inclusion body, and standing at room temperature until the inclusion body is dissolved; centrifuging at 4 ℃ and 10000r/min for 10 minutes, and taking a supernatant; adding 20% polyethylene glycol (PEG4000)210ul and 50mM oxidized glutathione 420ul, adding 100mM reduced glutathione 420ul, standing for 30min to 2h, dialyzing with PBS (pH8.0), and purifying with anti-GST affinity chromatography column, wherein the operation process strictly follows the instruction. Protein concentration determination a BCA protein quantification kit was used.
Example 2: antigenic characterization of VP1 protein and immunogenic polypeptides
Identification of antigenicity by Western Blot method:
adding the protein sample prepared in example 1 into 5 xSDS-PAGE loading buffer according to the ratio of 1:4, mixing uniformly, carrying out boiling water bath for 7min, centrifuging at 4 ℃ at 10000r/min for 2min, taking supernatant, carrying out electrophoresis, transferring the bacterial liquid protein onto a nitrocellulose membrane, and sealing with 5% skimmed milk powder for 40 min; the primary antibody is incubated with mouse-derived anti-EV 71 polyclonal antibody (1:5000) overnight at 4 ℃, the membrane is washed for 3 times by TBST, the secondary antibody is added with horseradish peroxidase-labeled goat anti-mouse IgG antibody (1:5000), the incubation is carried out for 30min at room temperature, and the membrane is washed for 3 times by TBST, and then DAB color development observation is carried out. Meanwhile, pET-49b (+) empty vector was transformed into DE3 E.coli induced expression product as negative control. The results are shown in FIGS. 6 to 10, respectively. As shown in the figure, the protein purification effect is better, and the purified proteins have antigenicity through identification.
Identification of antigenicity by ELISA:
the protein samples prepared in example 1 were coated on ELISA-coated plates, and two dilutions were made for each protein, so thatDiluting with coating buffer (coating buffer: containing Na in every 1L of distilled water)2CO3:1.59g,NaHCO32.93g), the protein concentration of the first dilution is: 2.08X 10-2mg/mL, second dilution protein concentration: 1.04X 10-2mg/mL; coating the protein and keeping the temperature at 4 ℃ overnight; washing the plate with PBST for 3 times 3min the next day; blocking with 5% Bovine Serum Albumin (BSA) at 37 ℃ for 1 h; PBST plate washing for 3 times, each time for 3 min; primary antibody is incubated for 2h at 37 ℃ with mouse-derived anti-EV 71 polyclonal antibody (1: 5000); PBST washing plate for 6 times, each time for 3 min; adding goat anti-mouse IgG antibody (1:5000) marked by horseradish peroxidase into the secondary antibody, and incubating for 1h at 37 ℃; PBST washing plate for 6 times, each time for 3 min; the TMB single-component color developing liquid develops color. Meanwhile, pET-49b (+) empty vector was transformed into DE3 E.coli induced expression product as negative control. The results are shown in Table 2.
TABLE 2 identification of the results of the detection of antigenicity by ELISA method
As shown in Table 2, with GST protein as a negative control, the A values of VP1 antigen and its immunogenic polypeptide increased more than 2 times compared with the A value of GST protein, and the result was identified as positive, i.e., the purified protein had antigenicity.
Example 3: identification of immunogenicity of VP1 proteins and immunogenic polypeptides
The VP1 protein and the immunogenic polypeptide purified in example 1 and Freund's adjuvant are mixed uniformly according to the volume ratio of 1:1, and then Balb/c mice are immunized, 5 mice are immunized by each protein, and the immunization is carried out for 4 times. 1.0mg of protein plus Freund complete adjuvant is immunized for the first time, 0.5mg of protein plus Freund incomplete adjuvant is immunized after 2 weeks, then 0.5mg of protein plus Freund incomplete adjuvant is continuously immunized for two times at intervals of 1 week, and the eyeball is picked and blood is taken after one week of last immunization; meanwhile, the pET-49b (+) empty vector expression protein is used for immunizing mice to take blood as a negative control. Western Blot was used to test whether sera reacted specifically with EV71 virus to test protein immunogenicity. The results are shown in FIG. 11.
As shown in the figure, EV71 virus cultured by RD cells is added into a loading hole of polyacrylamide gel, supernatant obtained by RD cell centrifugation is added into the loading hole as a negative control, and serum obtained after BALB/c mice are immunized by purified VP1 protein and polypeptide thereof is used as a primary antibody, wherein EV71 virus, VP1-GST fusion protein, VP179-432GST fusion protein, VP1436-864GST fusion protein, VP1436-735GST fusion protein and VP1565-864The serum obtained after mice are immunized with the GST fusion protein can specifically bind to the EV71 virus, and the molecular weight of the band shown in the figure is about 35KD (EV71 structural protein VP 1); serum obtained by immunizing mice with GST protein can also be specifically combined with EV71 virus, but the band shown in the figure has a molecular weight which is not EV71 structural protein VP 1; sera from PBS immunized mice did not bind specifically to EV71 virus (negative control).
SEQUENCE LISTING
<110> Shandong university
Shandong medical device quality inspection center
<120> immunogenic polypeptide of VP1 antigen of enterovirus 71 type, preparation method and application thereof
<130> 2015
<160> 20
<170> PatentIn version 3.5
<210> 1
<211> 297
<212> PRT
<213> SDLY107 strain VP1 amino acid sequence
<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> 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> 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> 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> 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. An immunogenic polypeptide of an enterovirus 71 VP1 antigen, which is characterized in that the amino acid sequence is shown as SEQ ID NO. 4.
2. A polynucleotide encoding the immunogenic polypeptide of claim 1, wherein the polynucleotide has the sequence shown in SEQ ID No. 9.
3. A recombinant vector comprising the polynucleotide of claim 2.
4. The recombinant vector according to claim 3, characterized in that its starting vector is pET-49b (+).
5. A recombinant expression bacterium comprising the recombinant vector according to claim 4.
6. The recombinant expression strain according to claim 5, wherein the starting strain is E.coli DE 3.
7. A method of producing the immunogenic polypeptide of claim 1, comprising the steps of:
(1) the gene fragment shown in SEQ ID NO.9 and a vector pET-49b (+) are subjected toSpeI andHandIII after double enzyme digestion, the expression vector pET-49b (+) -VP1 is obtained by connection construction436-735；
(2) The expression vector pET-49b (+) -VP1 of the step (1)436-735Transforming to Escherichia coli DE3, screening positive clone, and inducing to express recombinant protein;
(3) and (3) purifying the recombinant protein in the step (2) by adopting glutathione mercaptotransferase agarose gel to obtain the glutathione mercaptotransferase.
8. The method according to claim 7, wherein in the step (1), the gene fragment represented by SEQ ID NO.9 is constructed by: the full-length cDNA of SDLY107 is taken as a PCR template, and a primer sequence shown in SEQ ID NO. 17-SEQ ID NO.18 is taken as an amplification primer, and the cDNA is obtained by PCR amplification.
9. Use of the immunogenic polypeptide of claim 1 in the preparation of a diagnostic reagent for EV71 virus.
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| CN107449915A (en) * | 2017-07-17 | 2017-12-08 | 中国医学科学院医学生物学研究所 | A kind of method for detecting serum moderate resistance EV D68 antiviral antibodies |
| CN110079634A (en) * | 2019-03-21 | 2019-08-02 | 中国疾病预防控制中心病毒病预防控制所 | A kind of dual isothermal nucleic acid amplification method containing internal reference of quick detection enterovirus EV 71 type |
| CN113527442A (en) * | 2020-04-15 | 2021-10-22 | 南京蛋球球生物医学技术合伙企业(有限合伙) | Yolk antibody for inhibiting human enterovirus 71, preparation method and application thereof |
| CN113121657A (en) * | 2021-04-01 | 2021-07-16 | 新乡医学院 | Human enterovirus 71 and coxsackievirus A group 2VP1 recombinant protein, polyclonal antibody and application thereof |
| CN114989294A (en) * | 2022-05-27 | 2022-09-02 | 中国科学院武汉病毒研究所 | anti-EV 71 virus antibody and preparation method and application thereof |
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| CN108912213A (en) | 2018-11-30 |
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| CN105294843A (en) | 2016-02-03 |
| CN108912214A (en) | 2018-11-30 |
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