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Application of streptococcus pneumoniae protein to resisting infection of S. pneumoniae

A Streptococcus pneumoniae, protein technology, applied in the direction of immunoglobulin, antibacterial immunoglobulin, application, etc., can solve the problems of insufficient immunogenicity, adverse reactions, etc., to enhance resistance to Streptococcus pneumoniae infection, improve the body's IgG Effect of Antibody Response

Active Publication Date: 2019-03-12
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the above-mentioned shortcoming of the prior art, the object of the present invention is to provide the application of Streptococcus pneumoniae protein in the anti-Streptococcus pneumoniae infection, be used to solve its immunogenicity of most proteins in the prior art still not strong enough, can cause Adverse reactions and other issues

Method used

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  • Application of streptococcus pneumoniae protein to resisting infection of S. pneumoniae
  • Application of streptococcus pneumoniae protein to resisting infection of S. pneumoniae
  • Application of streptococcus pneumoniae protein to resisting infection of S. pneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Recombinant expression plasmid pET28a(+)-PepO, pET28a(+)-ZmpB 673-863 , pET28a(+)-ZmpB 673-863 -PepO and pET28a(+)-PepO-ZmpB 673-863 Construction of expression vectors.

[0089] (1) Materials:

[0090] Plasmid pET28a(+) was purchased from Novagen, Prime Star high-fidelity enzyme, dNTPs, Buffer, MgCl used in PCR 2 Purchased from Bao Biological Engineering (Dalian) Co., Ltd., PTC-200 PCR instrument was a Perkin Elmer product, and fluorescent quantitative PCR instrument RG-3000 was purchased from Corbett Research.

[0091] (2) Design and synthesis of primers:

[0092] Using Streptococcus pneumoniae D39 genomic DNA as a template, refer to its complete sequence (GeneBank number CP000410.2), use Premier5.0 to design primers, and synthesize them by Sangon Bioengineering (Shanghai) Co., Ltd.

[0093] ZmB 673-863 : Upstream primer: 5'-GCCATGGTTGAAGAAGTTGTTGTT-3', containing NcoI site;

[0094] Downstream primer: 5'-CCCTCGAGATCTCCAAGACTGTTAAT-3', containing XhoI site;

[...

Embodiment 2

[0162] Prokaryotic expression plasmids pET28a(+)-PepO, pET28a(+)-ZmpB 673-863 , pET28a(+)-ZmpB 673-863 -PepO and pET28a(+)-PepO-ZmpB 673-863 Expression, identification and purification in Escherichia coli.

[0163] (1) Recombinant plasmids pET28a(+)-PepO, pET28a(+)-ZmpB 673-863 , pET28a(+)-ZmpB 673-863 -PepO and pET28a(+)-PepO-ZmpB 673-863 Transformed into the host strain BL21(DE3).

[0164] (2) IPTG induces PepO and ZmpB 673-863 , ZmpB 673-863 -PepO (fusion protein) and PepO-ZmpB 673-863 (Fusion protein) expression in large quantities.

[0165] (3) Purification of the recombinant protein: After the bacteria were broken by ultrasonic, the supernatant of the broken bacteria was taken for purification; 4°C, 10000rpm×10min, the supernatant was filtered with a 0.45μm membrane filter, and the filtrate was collected for later use.

[0166] Affinity chromatography purification: pipette 2ml of 50% Ni 2+ -NTA resin suspension in the chromatography column, equilibrate with 20m...

Embodiment 3

[0180] Detection of ZmpB by Western blot 673-863 Recognition reaction of protein antiserum to Streptococcus pneumoniae bacterial protein.

[0181] (1) Streptococcus pneumoniae CMCC 31109 (type 1), D39 (type 2), CMCC 31436 (type 3), TIGR4 (type 4), CMCC 31207 (type 6B), CMCC31507 (type 7F), CMCC31216 (type 9V) , CMCC 31614 (Type 14), CMCC31687 (Type 18C), CMCC31693 (Type 19F) and CMCC31759 (Type 23F). A total of 11 different serotypes of bacteria were inoculated in 30ml C+Y medium, 37°C, 5% CO 2 Cultivate until the bacteria are in the logarithmic growth phase OD600=0.4-0.6, collect the bacteria, centrifuge at 12000g for 5min, wash twice with sterile PBS, resuspend the bacterial cell pellet with 100μl PBS, add 5×loadingbuffer in proportion, boil in boiling water for 30min, Centrifuge at 12000g for 5min, and take the supernatant for Western Blot analysis.

[0182](2) PAGE electrophoresis: the loading amount of protein per hole is 2 μg, 8% separating gel+5% stacking gel, 80V, 3...

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Abstract

The invention provides application of S. pneumoniae protein to resisting infection of S. pneumoniae. The endopeptidase O (PepO) of S. pneumoniae is a subcutaneous immunologic adjuvant, and the prepared S. pneumoniae protein vaccines have the good protection effects on resisting infection of S. pneumoniae through mixing and fusing expression of the subcutaneous immunologic adjuvant and 673rd to 863rd amino acid peptide fragment of zinc metal protease B (ZmpB).

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to Streptococcus pneumoniae PepO, ZmpB 673-863 Use in the fight against Streptococcus pneumoniae infection, especially related to the use of Streptococcus pneumoniae endopeptidase O as an adjuvant for subcutaneous immunization, ZmpB 673-863 Use as a vaccine alone in combination with other adjuvants and PepO and ZmpB 673-863 Application of mixed or fusion expression in anti-streptococcus pneumoniae infection. Background technique [0002] Streptococcus pneumoniae can cause pneumonia, meningitis and sepsis, and nearly 400,000 children under the age of 5 die every year worldwide due to Streptococcus pneumoniae infection. Pneumococcal sepsis is the leading cause of infant mortality in developing countries, where approximately 25% of deaths of children under 5 years of age and more than 1.2 million infant deaths are preventable each year. The current conjugate vaccine formulations...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/315C12N15/31C12N15/70C12N1/21C07K16/12A61K39/09A61P31/04
CPCA61K39/092A61P31/04C12N15/70C07K14/3156C07K16/1275A61K38/00
Inventor 尹一兵尹一帆张雪梅胥文春王虹何於娟肖江明王婧瑶
Owner CHONGQING MEDICAL UNIVERSITY
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