B-group epidemic neisseria meningitidis recombinant protein vaccine and preparing method thereof

A technology of epidemic meningitis and recombinant protein, which is applied in the direction of antibacterial drugs, pharmaceutical formulas, medical preparations containing active ingredients, etc., and can solve problems such as inappropriateness

Active Publication Date: 2015-09-09
BEIJING MINHAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, capsular polysaccharides from the surface of group B meningococci contain similar structures in mammalian and human embryonic tissues and gangliosides that may cross-react, so capsular polysaccharides from group B meningococci are not suitable for use in traditional meningococci In the research and development of polysaccharide vaccines or polysaccharide-protein conjugate vaccines against pneumococci, finding suitable non-capsular antigens is the key to developing group B meningococcal vaccines

Method used

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  • B-group epidemic neisseria meningitidis recombinant protein vaccine and preparing method thereof
  • B-group epidemic neisseria meningitidis recombinant protein vaccine and preparing method thereof
  • B-group epidemic neisseria meningitidis recombinant protein vaccine and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of Recombinant Protein Genetic Engineering Strain for Each Component of Group B Meningococcal Recombinant Protein Vaccine

[0037] 1. Construction of rHBP V1 engineering bacteria

[0038] According to the rHBP V1 coding gene published by NCBI (GenBank accession number: AE002098.2), the sequence was optimized according to the codon preference of Escherichia coli (SEQ ID No.1), and Bgl II and Xho I enzymes were added upstream and downstream of the sequence respectively cutting site; connect the synthetic rHBP V1 gene fragment with the expression vector pET-22b digested with the same endonuclease, introduce it into the host cell E.coli BL21(DE3), and obtain the high-efficiency rHBP V1 engineering project after screening Bacterial strain (expression level: 56.0mg / L), named: MH-rHBP V1 strain, and its preservation number is CGMCC NO.10718, and this bacterial strain has been preserved in China Microorganism Culture Preservation Management Committee Comm...

Embodiment 2

[0043] Example 2 Obtaining rHBP V1, rHBP V2, rHBP V3 recombinant proteins

[0044] The three variants of rHBP, rHBP V1, rHBP V2, and rHBP V3, were obtained through the following steps: seed solution preparation, fermentation, centrifugation to collect cells, cell crushing, filter sterilization, and cryopreservation. The following is the method for obtaining rHBP V1, and the methods for obtaining rHBP V2 and rHBP V3 refer to rHBP V1.

[0045] First inoculate the rHBP V1 working seed batch bacterial strain (MH-rHBP V1 strain, whose preservation number is CGMCC NO.10718) obtained in Example 1 into fresh LB liquid medium containing ampicillin with a final concentration of 50 μg / mL, 37 Cultivate overnight at ℃ to prepare seed liquid, which is used for inoculation of fermenter tanks, and the medium suitable for the growth of Escherichia coli can be selected for fermentation culture. Fermentation parameters: 37°C, pH7.0-7.2, 200 rpm; when the bacteria grow to OD 600 When =15, add I...

Embodiment 3

[0046] Example 3 Preparation of Antigen and Aluminum Hydroxide Adjuvant Adsorption Product and Preparation of Group B Meningococcal Recombinant Protein Vaccine

[0047] The preparation method of aluminum hydroxide adjuvant is: in PBS, slowly add 10% KAl (SO 4 ) 2 , magnetic stirrer, as long as no vortex is formed, mix well. Slowly add 0.5mol / L NaOH dropwise to the mixture until the pH reaches 7.0. After standing at 4°C for 24 hours, replace the medium with 0.85% physiological sodium chloride solution, and dilute to the original volume. The solution was changed four times with an interval of 8 h between each time. After each liquid change, stir for 30 minutes and place in a refrigerator at 4°C. For the last liquid change, use NaOH to adjust the pH to 6.0-7.0, and treat with high pressure before use.

[0048] 1. Preparation of adsorption product of rHBP V1 and aluminum hydroxide adjuvant

[0049] Take the sterilized and filtered rHBP V1 protein prepared in Example 2, adjus...

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Abstract

The invention provides a B-group epidemic neisseria meningitidis recombinant protein vaccine and a preparing method thereof, and belongs to the field of biological products. The vaccine contains three mutant recombinant proteins of a human H factor binding protein. The invention further provides a preparing method of the B-group epidemic neisseria meningitidis recombinant protein vaccine. The method includes the steps of human H factor binding protein V1, V2 and V3 recombinant expression carrier construction, expression strain screening and identification, fermental cultivation, recombinant protein antigen inducible expression and recombinant protein antigen purification and protein detection; and according to the concentrations of the three antigen components, quantified mixing is carried out, and the B-group epidemic neisseria meningitidis recombinant protein vaccine is prepared in cooperation with an aluminum adjuvant. By means of the B-group epidemic neisseria meningitidis recombinant protein vaccine, intraperitoneal injection is carried out on an immune mice, the mice body can be stimulated to generate a B-group epidemic neisseria resisting peculiar antibody, and the alexin mediated serum sterilization antibody can rise by more than 8 times.

Description

technical field [0001] The invention relates to the field of biological products, in particular to a group B meningococcal recombinant protein vaccine and a preparation method thereof. Background technique [0002] Neisseria meningitidis is the main pathogenic bacterium of bacterial meningitis and sepsis. According to the differences in the structure and antigenicity of the capsular polysaccharide on the surface of meningococci, meningococci are divided into 13 serogroups (Serogroup) , of which 5 groups (A, B, C, W135 and Y groups) are the main pathogenic bacteria groups. The global incidence of meningitis caused by meningococcus reaches 300,000-500,000 per year, and about 50% of the infected people are mainly caused by group B meningococcus. In recent years, due to the abuse of antibiotics, drug-resistant strains have emerged around the world, which has added greater difficulties to the treatment of diseases. [0003] It is generally believed that inoculation of safe and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/095A61P31/04
Inventor 林福玉魏文进孟夏萌任玉莹张静飞郑海发
Owner BEIJING MINHAI BIOTECH
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