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Meningococcus antigen combination and application thereof

An antigen and vaccine composition technology, applied in the field of vaccines, can solve the problems of increasing the titer and side effects of vaccines and the like

Active Publication Date: 2014-10-01
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether there is a titer interaction between various antigens in multi-antigen vaccines and whether it will cause increased side effects are potential risks in the vaccine preparation process.

Method used

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  • Meningococcus antigen combination and application thereof
  • Meningococcus antigen combination and application thereof
  • Meningococcus antigen combination and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1 Construction of Recombinant Expression Engineering Bacteria

[0095] Genomes were extracted from the cultures of three clinical isolates of meningococcus, and the first fHbp (named fHbp-1, ie variant 1) and the second fHbp ( Named as fHbp-2, namely variant 2) and NHBA gene. PCR amplification forward primer and reverse primer are listed in the table below, and forward primer and reverse primer introduce the restriction enzyme cutting site of Nde I and BamH I (fHbp) or Nhe I and BamH I (NHBA) respectively, with are underlined. The recombinant fHbp and NHBA proteins were expressed in Escherichia coli in the form of His-tag fusion, and the plasmid pET-28a (Novagen) was used as the expression vector.

[0096]

[0097] Cloning of fHbp-1 and fHbp-2: PCR was performed from bacterial genomic DNA using rTaq DNA polymerase (TaKaRa). The PCR reaction conditions were: 95°C for 5 min; 35 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min; 72°C for 8 min. Th...

Embodiment 2

[0101] Embodiment 2 fermentation culture

[0102] Fermentation of recombinant fHbp-1 and fHbp-2. Transform the recombinant plasmid into BL21 (DE3) competent, pick the monoclonal colony on the plate, inoculate it into 5ml LB liquid medium containing kanamycin, culture it with shaking at 37°C overnight, and transfer it to 250ml kanamycin-containing medium the next day In the Erlenmeyer flask of the LB liquid medium of mycin, shake culture at 37°C until the absorbance value of 600nm is A 600 value is 0.6, add IPTG to a final concentration of 0.6mmol / L, continue shaking culture for 5 hours, centrifuge at 6000rpm for 10 minutes, and collect the bacteria.

[0103] Fermentation of recombinant NHBA. Transform the recombinant plasmid into BL21 (DE3) competent, pick the monoclonal colony on the plate, inoculate it into 5ml LB liquid medium containing kanamycin, culture it with shaking at 37°C overnight, and transfer it to 250ml kanamycin-containing medium the next day In the Erlenmey...

Embodiment 3

[0104] Example 3 Purification of target protein

[0105] Resuspend the bacterial body fluid with nickel column equilibration buffer and place it in ice-water mixture, and ultrasonically break the bacteria. After breaking the bacteria, the supernatant was harvested by centrifugation. Ni 2+ The crude product was purified by affinity chromatography column, the recombinant fHbp-1 and fHbp-2 were further purified by gel filtration chromatography, and the recombinant NHBA was further purified by CM ion column. The SDS-PAGE electrophoresis patterns of purified recombinant fHbp-1, fHbp-2 and NHBA proteins are shown in Figure 4 . The purified protein was stored at -20°C for future use.

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Abstract

The invention provides a novel antigen combination of meningococcus and an application thereof. The antigen combination comprises fHbp V1 variant proteins, fHbp V2 variant proteins and NHBA proteins. The experiment shows that vaccines prepared from the antigen combination have an obvious synergistic effect and have high broad-spectrum meningococcus resistant capability.

Description

technical field [0001] The invention relates to the vaccine field, in particular to meningococcal recombinant protein vaccine components and combinations thereof. Background technique [0002] Bacterial meningitis is a serious threat to human health, with an estimated 170,000 deaths worldwide each year, and Neisseria meningitidis is one of the three main pathogenic microorganisms that cause bacterial meningitis, and is the only one that causes epidemic meningitis ( Meningitis) pathogenic bacteria. The average incidence of meningococcal meningitis in the world is 1-10 / 100,000, the case fatality rate is about 10%, and more than 20% of patients will leave permanent damage to the central nervous system. [0003] According to the different chemical structures of capsular polysaccharides, meningococcus can be divided into 13 serogroups, and the main pathogenic bacteria groups are A, B, C, Y and W135 groups, which can cause more than 95% of cases. Other serogroups such as 29E and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K16/12C12N15/31C12N15/63C12N5/10C12N15/70C12N1/21A61K39/095A61K38/16A61P31/04
CPCA61K39/00A61K39/095C07K14/22Y02A50/30
Inventor 朱为吴根鹏袁萍黄帼英王晓毕慧荣家康
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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