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Streptococcus suis △cps/ssna-msly(p353l)-sc19 engineering bacteria and its application in vaccines

A-SC19, Streptococcus suis technology, applied in the field of veterinary biological products, can solve the problems of high cost, low immune efficacy, strong virulence, etc., and achieve the effects of reducing side effects, improving safety, and reducing side effects

Active Publication Date: 2021-07-27
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has achieved certain effects, it is subject to many restrictions such as high cost, low immune efficacy and the risk of strong virulence in actual application.

Method used

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  • Streptococcus suis △cps/ssna-msly(p353l)-sc19 engineering bacteria and its application in vaccines
  • Streptococcus suis △cps/ssna-msly(p353l)-sc19 engineering bacteria and its application in vaccines
  • Streptococcus suis △cps/ssna-msly(p353l)-sc19 engineering bacteria and its application in vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] 1. △CPS / SsnA-mSly(P353L)-SC19 engineering bacteria

[0102] Specific construction strategies such as figure 1 As shown, the construction method includes the following steps:

[0103] 1) According to the ORF sequence of the cpsEF gene in the whole genome sequence of Streptococcus suis type 2 05ZYH33 strain (GenBank: CP000407.1) published on NCBI such as SEQ ID No.1, the ORF sequence of the ssna gene is shown as SEQ ID No. 2 and the ORF sequence of the sly gene, such as SEQ ID No.3, design primers for knocking out the cpsEF gene and the ssna gene, and construct a primer pair for the point mutation of the sly gene, respectively:

[0104] a. Construction of primers for knocking out the cpsEF gene:

[0105] Upstream homology arm forward and reverse primers:

[0106] ECORI-E-1: AAAGAATTCGGCTCGTGCTATATTCTCTTGG,

[0107] RONGHE-EF-2:GAATCTTTTTCATAACAGCTCTCTCCACTATTTC;

[0108] Downstream homology arm forward and reverse primers:

[0109] RONGHE-EF-1:

[0110] GAAATAGTGGA...

Embodiment 2

[0230] Example 2: The safety of the inactivated vaccine prepared with △CPS / SsnA-mSly(P353L)-SC19 and the immune protection effect to multiple serotype Streptococcus suis infection

[0231] 1. Preparation of inactivated vaccine using △CPS / SsnA-mSly(P353L)-SC19 strain as antigen

[0232] (1) Bacterial solution preparation

[0233] Pick △CPS / SsnA-mSly(P353L)-SC19 from the plate and culture it overnight in TSB (containing 5% serum), and then transfer the bacteria cultured to the stationary phase to 300ml TSB (containing 5% serum) at a ratio of 1:100. % serum), cultured for 12 hours.

[0234] (2) Bacterial antigen inactivation

[0235] Bacteria liquid that has been cultivated to the stable period: take out 1ml for bacterial purity detection and counting, and conduct inspection according to the appendix of the current "Chinese Veterinary Pharmacopoeia". The result shows that the number of bacteria in each ml of bacterial liquid is ; the remaining bacteria are inactivated by addin...

Embodiment 3

[0257] Example 3: The safety of the attenuated vaccine prepared with △CPS / SsnA-mSly(P353L)-SC19 and the immune protection effect to multiple serotypes of Streptococcus suis infection

[0258] 1. Safety test of △CPS / SsnA-mSly(P353L)-SC19 as an attenuated vaccine strain

[0259] Pick a single colony of the wild strain SC19 and the mutant strain △CPS / SsnA-mSly(P353L)-SC19 from the fresh plate cultured overnight, place it in 5ml TSB (containing 5% serum) and place it at 37 degrees at 160 rpm for 12 hours, and a blank control was set. During the cultivation process, 100 μl was taken out from each group every hour to measure the light absorption value at OD600, and the growth curve was drawn. The experiment was repeated three times, and it was observed that there was no significant difference in the growth rate between the wild strain and the mutant, indicating that the mutation did not affect the growth characteristics of Streptococcus suis in the medium. Bacterial counts in the ...

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Abstract

The invention discloses a Streptococcus suis △CPS / SsnA-mSly(P353L)-SC19 engineering bacterium and its application in vaccines. The strain uses the virulent isolate strain SC19 of Streptococcus suis type 2 as the starting strain, and adopts the method of homologous recombination , knocked out the virulence genes cps and ssna, and mutated the 353 amino acids of the hemolysin gene from P to L, and successfully constructed △CPS / SsnA‑mSly(P353L)‑SC19. The pathogenicity of the strain △CPS / SsnA‑mSly(P353L)‑SC19 is significantly reduced, and it has good safety when used as an inactivated vaccine and an attenuated vaccine strain. The protective efficacy of infection with other serotype strains. Moreover, the vaccine will not produce antibodies against SsnA after immunization, and this strain can be used as a strain of gene-marked vaccine to distinguish vaccine-immunized animals from wild-virus-infected animals, thereby facilitating the eradication and purification of the disease.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, in particular to a Streptococcus suis ΔCPS / SsnA-mSly(P353L)-SC19 engineering bacterium and its application in vaccines. Background technique [0002] Streptococcus suis is a zoonotic acute febrile infectious disease caused by a variety of pathogenic streptococci. The main pathogen is Streptococcus suis, which includes many serotypes, 33 of which have been typed, and some strains that cannot be typed. There is a lack of cross-immune protection among the serotypes. As a result, the inactivated vaccines currently on the market are usually only aimed at specific serotypes, but cannot prevent the infection of multiple serotypes. Therefore, it is of great value and significance to develop a wide range of vaccines capable of preventing infection by multiple serotypes of Streptococcus suis. [0003] Existing research results show that the difference of serotype mainly depends on ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74A61K39/09A61P31/04C12R1/46
CPCA61K39/092A61K2039/521A61K2039/522A61P31/04C07K14/315C12N9/22
Inventor 张安定徐磊林岚吕伟华韩丽
Owner HUAZHONG AGRI UNIV
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