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Meningococcus antigen composition and applications thereof

A meningitis and antigen technology, applied in the field of vaccines, can solve the problems of vaccines with increased potency and side effects

Active Publication Date: 2017-11-17
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 composition and applications thereof
  • Meningococcus antigen composition and applications thereof
  • Meningococcus antigen composition and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 Construction of recombinant expression engineering bacteria

[0096] The genomes were extracted from the cultures of three clinical isolates of meningococcus, and the first fHbp (named fHbp-1, namely variant 1) and the second fHbp ( Named fHbp-2, which is variant 2) and NHBA gene. The PCR amplification forward primer and reverse primer are listed in the table below. The forward primer and reverse primer introduce the restriction enzyme sites of Nde I and BamH I (fHbp) or Nhe I and BamH I (NHBA), respectively. The underlined display. Recombinant fHbp and NHBA proteins were expressed in E. coli in the form of His-tag fusion and plasmid pET-28a (Novagen) was used as the expression vector.

[0097]

[0098] Cloning of fHbp-1 and fHbp-2: PCR was performed from bacterial genomic DNA with rTaq DNA polymerase (TaKaRa). The PCR reaction conditions were: 95°C for 5 min; 95°C for 1 min, 60°C for 1 min, 72°C for 1 min, 35 cycles; 72°C for 8 min extension. The amplified pro...

Embodiment 2

[0102] Example 2 Fermentation culture

[0103] Fermentation culture 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 with shaking at 37℃ overnight, and transfer 250ml containing kanamycin the next day In an Erlenmeyer flask of LB liquid medium, culture with shaking at 37℃ to 600nm absorbance value A 600 The 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.

[0104] Fermentation culture 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 with shaking at 37℃ overnight, and transfer 250ml containing kanamycin the next day In an Erlenmeyer flask of LB liquid medium, culture with shak...

Embodiment 3

[0105] Example 3 Purification of target protein

[0106] Resuspend the bacterial body fluid with a nickel column equilibration buffer and place it in a mixture of ice and water, and ultrasonically break the bacteria. After sterilization, 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. See the SDS-PAGE electrophoresis pattern of the purified recombinant fHbp-1, fHbp-2 and NHBA protein Figure 4 . The purified protein is stored at -20°C for later use.

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Abstract

The invention provides a novel meningococcus antigen composition and applications thereof. Specifically, the antigen composition comprises fHbp V1 mutant protein, fHbp V2 mutant protein, and NHBA protein. The experiment results show that a vaccine prepared from the antigen composition has a prominent synergistic effect and an excellent performance on resisting meningitis in a broad spectrum.

Description

[0001] This application is a divisional application for an invention patent application with an application date of June 24, 2014, an application number of 201410290161.9, and an invention title of "a meningococcal antigen combination and its application". Technical field [0002] The present invention relates to the field of vaccines, in particular to the components and combinations of meningococcal recombinant protein vaccines. Background technique [0003] Bacterial meningitis seriously threatens human health. There are an estimated 170,000 deaths worldwide each year. Neisseria meningitis is one of the three main pathogenic microorganisms that cause bacterial meningitis and the only cause of epidemic meningitis ( Meningococcal) pathogenic bacteria. The global average incidence of meningococcal meningitis is 1-10 / 100,000, and the fatality rate is about 10%. More than 20% of patients will have permanent central nervous system damage. [0004] According to the chemical structure of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/095A61P31/04
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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