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Neisseria meningitidis vaccine and preparation method and application thereof

A meningococcal and vaccine technology, applied in the field of biomedicine, can solve sensitivity and other problems, and achieve the effect of broad antigen spectrum and high bactericidal activity titer

Active Publication Date: 2020-02-18
苏州微超生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nearly 12 anti-fHBP monoclonal antibodies (MAbs) have been studied, and most of the MAbs can significantly inhibit the binding of fH to fHBP or the bacterial surface, thus making the bacteria more sensitive to lysis mediated by the alternative pathway

Method used

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  • Neisseria meningitidis vaccine and preparation method and application thereof
  • Neisseria meningitidis vaccine and preparation method and application thereof
  • Neisseria meningitidis vaccine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0038] The preparation method of the fusion protein of the present invention is characterized in that it comprises the following steps:

[0039] Constructing a recombinant plasmid comprising the coding gene as described above;

[0040] Transforming the recombinant plasmid into host cells, identifying, screening and identifying correct positive strains, and transforming the correctly identified positive strains into expression cells; culturing.

[0041] Preferably, the recombinant plasmid containing the above-mentioned coding gene is obtained by inserting the sequence of the coding gene into a plasmid vector by double restriction digestion. More preferably, the vector is pET32a(+).

[0042] Preferably, the expression strain is E. coli BL21(DE3).

[0043] The group B meningococcal vaccine of the present invention includes the fusion protein as described in any one of the above.

[0044] Preferably, the fusion protein includes: chimeric protein I and chimeric protein II; the ami...

Embodiment 1

[0064] 1. Design of group B meningococcal fHBP chimeric protein

[0065] The N-terminal partial sequence of V1 (including structural domain A, B and partial structural domain C, B01 ABpC ) and the partial sequence of domain C of V2 (partial domain C, A19 pC ) for fusion, and the Linker between them is a small peptide segment (GEHT) in the amino acid sequence of fHBP, namely B01 ABpC -A19 pC . Since there is no similarity sequence between these two fragments, they can be connected by overlapping PCR method (such as figure 1 shown).

[0066] then B01 ABpC -A19 pC As templates, two chimeric proteins (chimeric protein I: Cp-1 and chimeric protein II: Cp-2) were constructed by gene site-directed mutagenesis, as shown in Table 1 below.

[0067] Table 1 Site-directed mutation information of two chimeric proteins

[0068]

[0069] 2. Technical methods

[0070] 1) PCR amplifies three variant genes respectively:

[0071] The N-terminal partial sequence of V1 variant B01 (B0...

Embodiment 2

[0147] Example 2 Vaccine preparation and immune effect test

[0148] Vaccine preparation: under sterile conditions, the chimeric protein I (Cp-1) and chimeric protein II (Cp-2) described in Example 1 were mixed with equimolar mass, and then mixed with Freund's adjuvant ( Complete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for the second immunization) were mixed at a volume ratio of 1:1.

[0149] Contrast vaccine: equal amounts of variant V1 protein and variant V2 protein (amino acid sequences are respectively SEQ ID NO.26-27) were mixed with Freund's adjuvant at a volume ratio of 1:1, and two equal amounts of variable The total molar mass of the body protein was equal to the total molar mass of chimeric protein I and chimeric protein II, and the ultrasonic emulsification was carried out under ice bath conditions.

[0150] SEQ ID NO.26[V1, B01, mature peptide]

[0151] MSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKL...

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Abstract

The invention provides neisseria meningitidis fHBP changeable body fusion protein. The amino acid sequence of the protein is formed through sequentially connecting the amino acid sequence of an fHBP variant V1 with the amino acid sequence of an fHBP variant V2 and the amino acid sequence of an fHBP variant V3 through connexons, and the fusion protein containing the polypeptide segments is prepared. The protein is easy to express and can be rightly folded. Compared with a vaccine prepared from protein of any single variant of a variant V1, a variant V2 and a variant V3 or mixed protein of the three variants, a vaccine prepared from the fusion protein has better immunogenicity, after animals are immunized, an antibody having higher titer can be obtained, and a spectrotype is wider than thatof a conventional vaccine and can cover more kinds of variant strains.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, in particular to a group B meningococcal vaccine and its preparation method and application. Background technique [0002] Group B meningococci (Neisseria. meningitidis, MenB) are different from the capsular polysaccharides of the other 4 species of meningococci (A, C, W135 and Y). Studies have shown that the capsular polysaccharide of MenB is similar to the sugar on the surface of many human cells (such as cells during fetal brain development). This capsular polysaccharide is α(2→8)N-acetylneuraminic acid (also known as polysialic acid ) homopolymer. Therefore, the capsular polysaccharide of MenB is recognized as an autoantigen in humans and has poor immunogenicity, which is not suitable as an effective component for MenB vaccine development. [0003] Current vaccine development for MenB has focused on non-capsular polysaccharide immunogens, especially membrane proteins or outer membrane v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/095A61K39/39A61P31/04C12R1/19
CPCA61K39/095A61K39/39A61K2039/55566A61P31/04C07K14/22C07K2319/40C12N15/70
Inventor 胡浩陈瑞勤蒋浩然于旭博
Owner 苏州微超生物科技有限公司
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