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Immunogenic polypeptide of 71-type VP1 antigen of enterovirus, preparation method and application thereof

A technology of immunogenic polypeptides and enteroviruses, applied in the field of immunobiology, to achieve the effects of simple operation, simple expression and purification methods, good antigenicity and immunogenicity

Active Publication Date: 2018-11-30
SHANDONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The BC loop region (91-106 amino acid sites) of VP1 protein is currently recognized as the site of neutralizing epitopes, but whether there are other important epitopes still needs further research and demonstration

Method used

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  • Immunogenic polypeptide of 71-type VP1 antigen of enterovirus, preparation method and application thereof
  • Immunogenic polypeptide of 71-type VP1 antigen of enterovirus, preparation method and application thereof
  • Immunogenic polypeptide of 71-type VP1 antigen of enterovirus, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Expression and purification of the immunogenic polypeptide of the VP1 antigen of enterovirus 71

[0054] 1. Amplify the cDNA of EV71 SDLY107 strain virus

[0055] Use OMEGA's Viral RNA kit to extract the RNA of SDLY107 strain virus. The virus strain SDLY107 was isolated from a child with hand, foot and mouth disease who died in Linyi People's Hospital of Shandong Province. Fast and strong, with obvious animal pathological characteristics), the strain has been subjected to full sequence analysis (GenBank: JX244186.1), and cDNA was generated by reverse transcription using TOYOBO's ReverTra Ace qPCR Kit.

[0056] 2. Primer design and PCR amplification

[0057] The full-length cDNA of SDLY107 was used as a PCR template to design primers for PCR amplification. The primer sequences are shown in Table 1:

[0058] Table 1 Primer Sequence

[0059]

[0060] With SDLY107 full-length cDNA as PCR template, SpeI VP1 Primer F is upstream primer, and HindIII VP1Primer ...

Embodiment 2

[0068] Example 2: Antigenic Identification of VP1 Protein and Immunogenic Polypeptides

[0069] 1. Identification of antigenicity by Western Blot method:

[0070] Add the protein sample prepared in Example 1 to 5×SDS-PAGE loading buffer at a ratio of 1:4, mix well, then boil in water bath for 7 minutes, centrifuge at 10,000 r / min at 4°C for 2 minutes, take the supernatant for electrophoresis, and transfer the bacterial liquid protein On the nitrocellulose membrane, 5% skimmed milk powder was blocked for 40min; the primary antibody was incubated with mouse-derived anti-EV71 polyclonal antibody (1:5000) at 4°C overnight, and the membrane was washed 3 times with TBST, and the secondary antibody was added with horseradish peroxidase Labeled goat anti-mouse IgG antibody (1:5000), incubated at room temperature for 30 minutes, washed the membrane three times with TBST, and added DAB for color development. At the same time, the pET-49b (+) empty vector was transformed into DE3 Escher...

Embodiment 3

[0077] Example 3: Identification of Immunogenicity of VP1 Protein and Immunogenic Polypeptide

[0078] The VP1 protein and immunogenic polypeptide purified in Example 1 were mixed with Freund's adjuvant at a ratio of 1:1 by volume, and then Balb / c mice were immunized, and 5 mice were immunized for each protein, and immunized 4 times in total. 1.0 mg protein plus Freund's incomplete adjuvant for the first immunization, 0.5 mg protein plus Freund's incomplete adjuvant after 2 weeks, then 0.5 mg protein plus Freund's incomplete adjuvant for two consecutive immunizations at intervals of 1 week, and the last immunization week Afterwards, the eyeballs were removed and blood was taken; at the same time, blood was taken from mice immunized with pET-49b(+) empty vector expressing protein as a negative control. Western Blot was used to detect the specific reaction between serum and EV71 virus to detect protein immunogenicity. The result is as Figure 11 shown.

[0079] As shown in th...

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Abstract

The invention belongs to the field of immunobiology and relates to immunogenic polypeptide of 71-type VP1 antigen of enterovirus, a preparation method and application thereof, and particularly relatesto the immunogenic polypeptide of the 71-type VP1 antigen of the enterovirus. The immunogenic polypeptide 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 formed by substituting, deleting or adding one or more amino acids for the sequence and with equal functions. The invention also relates to the preparation method of theimmunogenic polypeptide and the application to preparation of a diagnostic reagent for EV71 viruses. The immunogenic polypeptide has good antigenicity and immunogenicity and provides research basis for early development of the diagnostic reagent and vaccine research and development of EV71.

Description

technical field [0001] The invention belongs to the technical field of immunobiology, and in particular relates to an immunogenic polypeptide of enterovirus 71 type VP1 antigen, a preparation method and application thereof. Background technique [0002] EV71 is a single-stranded positive-sense RNA virus belonging to the genus Enterovirus A of the Picornaviridae family. Its infection can cause hand, foot and mouth disease in infants and young children. The main manifestations of the disease are fever, diarrhea and herpetic angina. Different from typical HFMD, infection with certain EV71 strains can cause severe HFMD, with complications including aseptic meningitis, encephalitis, polio-like paralysis, neurogenic pulmonary edema and even death. The incidence of EV71 infection is widespread, and its epidemic range spreads to Asia, Europe, North America, Australia, etc., and the mortality rate is high. It is a public health problem that is currently receiving considerable attenti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/085C12N15/41C12N15/70C12N1/21G01N33/68G01N33/569
CPCC07K14/005C12N2770/32322G01N33/56983G01N33/68G01N2333/085
Inventor 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
Owner SHANDONG UNIV
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