Serogroup B meningococcus recombinant chimeric protein vaccine and preparation method thereof

A chimeric protein, meningococcal meningitis technology, applied in the field of group B meningococcal recombinant chimeric protein vaccine and its preparation, can solve the problem of lack of immunogenicity, achieve effective vaccine protection effect, increase preventive effect, Enhanced immunogenicity

Active Publication Date: 2018-03-23
BEIJING ZHIFEI LVZHU BIOPHARM +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the linear polysialic acid contained in the capsular polysaccharide of group B Nm is similar to the structure of human N-acetylneuraminic acid polymer, so it

Method used

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  • Serogroup B meningococcus recombinant chimeric protein vaccine and preparation method thereof
  • Serogroup B meningococcus recombinant chimeric protein vaccine and preparation method thereof
  • Serogroup B meningococcus recombinant chimeric protein vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1P2-f

[0056] The construction of embodiment 1P2-fHBP V1 engineering strain:

[0057] According to the amino acid sequence of fHBP V1 and P2 and the codon preference of Escherichia coli, the full-length DNA sequence p2-fhbp V1 was designed and synthesized, and p2 was connected to the N-terminus of fhbp V1 as a gene synthesis plasmid, and the gene p2-fhbp V1 was synthesized into plasmid pUC57 Above, the gene synthesis plasmid pUC57 and the vector pET43.1a were digested with restriction enzymes NdeI and XhoI to obtain the target gene fragment and the digested plasmid pET43.1a; the target gene fragment p2-fhbp V1 was combined with the plasmid pET43. 1a was ligated with T4ligase, and the ligated product was transformed into E.coli DH5α, and cultured overnight at 37°C. Pick positive clones to inoculate LB liquid medium, shake culture at 37°C overnight. The target gene fragment was amplified by PCR method, and the analysis and identification results were as follows: figure 1 It is shown ...

Embodiment 2P2-f

[0059] The fermentation of embodiment 2P2-fHBP V1 engineering bacteria

[0060] The strains were inoculated into LB liquid medium, and cultured at 37° C. and 200 rpm for 12 hours. Expand the inoculation to the fermenter at a ratio of 5%, culture at 37°C for 4-8 hours, and when the OD600 increases to 18-22, induce with IPTG at a final concentration of 0.05mM for 4-8 hours. Bacteria were collected by centrifugation in a large-capacity centrifuge.

Embodiment 3P2-f

[0061] Extraction and purification of embodiment 3P2-fHBP V1 recombinant chimeric protein

[0062] Bacterial destruction: Take the bacterial cells and add the bacterial destruction buffer (20mM PB, 2mM EDTA, pH7.0), homogeneously destroy the bacteria twice at low temperature (4°C) and ultra-high pressure (1100-1400bar), then centrifuge at a high speed (10000rpm, 60min , 4°C) to take the broken supernatant of bacteria.

[0063] Protein extraction: using the method of ammonium sulfate fractional precipitation, 15% ammonium sulfate was precipitated at 4°C for 1 hour, centrifuged at 6500rpm at 8°C for 60 minutes, and the centrifuged supernatant was taken; the supernatant was stirred and precipitated with 45% ammonium sulfate at 4°C for 1 hour, The protein precipitate was dissolved in 20mM PB pH7.4, ultrafiltered with a 10KD membrane bag and concentrated to 1 / 5-1 / 3 of the original volume.

[0064] Refining protein purification: Sepharose QFF was used to load the sample with 20mM P...

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Abstract

The invention provides a serogroup B meningococcus recombinant chimeric protein vaccine and a preparation method thereof. The vaccine contains three recombinant chimeric proteins of P2-fHBP V1, P2-fHBP V2 and P2-fHBP V3, wherein amino acid sequences of the recombinant chimeric proteins are SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; the contents of all the proteins are 30 to 50mu g/dose; optimally, a vaccine preparation also contains a cryoprotectant or vaccine adjuvant. The invention also provides the preparation method of the three recombinant chimeric proteins. By using the recombinant chimeric proteins formed by linking P2 with serogroup B meningococcus fHBP protein, humoral immune response can be effectively induced, and immunogenicity of fHBP is obviously improved. The vaccine provided by the invention can effectively cover all serogroup B meningococcus strains, thereby providing a broad-spectrum preventing effect on invasive diseases such as cerebrospinal meningitis,bacteremia, pneumonia and pericarditis caused by the serogroup B meningococcus.

Description

technical field [0001] The present invention relates to the preparation of a vaccine, in particular to a group B meningococcal recombinant chimeric protein vaccine and a preparation method thereof, wherein the group B meningococcal recombinant chimeric protein vaccine comprises group B meningococcal human H factor combined Protein (fHBP) is a recombinant chimeric protein formed by linking tetanus universal T cell epitope P2. The protein has three variants fHBPV1, fHBP V2 and fHBP V3, and the three variant recombinant chimeric proteins are prepared in proportion Vaccine formulations and methods for their preparation. Background technique [0002] Neisseria meningitidis (Nm), commonly known as meningococcus (Meningococcus), can cause epidemic cerebrospinal meningitis (referred to as meningitis) and sepsis, and the disease has a high morbidity and mortality. Neisseria meningitidis is a pathogenic Gram-negative diplococcus with humans as its main host. It has caused outbreaks a...

Claims

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Application Information

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IPC IPC(8): A61K39/095A61P31/04A61P25/00C07K19/00C12N15/70
CPCA61K39/095A61K2039/5158A61K2039/70C07K14/22C07K2319/40C12N15/62C12N15/70C12N2800/22
Inventor 苏桂民朱卫华高博郭通纪国存冀颖杜琳
Owner BEIJING ZHIFEI LVZHU BIOPHARM
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