Streptococcussuis serotype 2 SsnA gene knockout mutant strain, and preparation method and application of mutant strain

A technology of Streptococcus suis and gene knockout, applied in the field of genetic engineering, can solve problems such as unclear biological functions

Inactive Publication Date: 2015-05-13
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

till this moment SsnA Biological function of gene in Streptococcus suis unknown

Method used

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  • Streptococcussuis serotype 2 SsnA gene knockout mutant strain, and preparation method and application of mutant strain
  • Streptococcussuis serotype 2 SsnA gene knockout mutant strain, and preparation method and application of mutant strain
  • Streptococcussuis serotype 2 SsnA gene knockout mutant strain, and preparation method and application of mutant strain

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Experimental program
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Effect test

Embodiment 1

[0039] The embodiment isolates and identifies type 2 Streptococcus suis wild-type SS2-GD01 strain (SsnA + ) (preserved in China Center for Type Culture Collection (CCTCC) on October 31, 2014, address: China, Wuhan, Wuhan University, deposit number is CCTCC NO: M2014538) as the parent strain, against SsnA Specific primers were designed for the upstream and downstream sequences of the gene to amplify the upstream and downstream homology arm sequences; the chloramphenicol resistance gene cassette was amplified by PCR using the shuttle plasmid pSET3 as a template, and three fragments were constructed by enzyme-cut ligation to construct the suicide The sex vector plasmid pSET4SΔSsnA was transformed into the parental strain by electroporation, and finally obtained by PCR screening and identification SsnA Gene knockout mutant strain (SS2-GD01ΔSsnA). Specific steps are as follows:

[0040] 1, SsnA Amplification of upstream and downstream sequences of genes

[0041] According to...

Embodiment 2

[0077] Genetic Stability Analysis of Gene Knockout Mutant SS2-GD01ΔSsnA

[0078] Prepared by Example 1 SsnA Knockout mutant strain SS2-GD01ΔSsnA in TSA Cm R Streak culture was carried out on the plate, and a single colony was picked and inoculated into TSB medium, and cultured at 37°C and 200 r / min for 12 h, and the proliferated bacterial liquid was 1 generation. Then the first-generation bacterial solution was streaked on the TSA plate, and a single colony was picked and inoculated into the TSB medium, and cultured with shaking at 37°C for 12 h. Subculture, continuous culture for 20 generations, PCR amplification and identification of the proliferated bacterial liquid with SsnA-JD-F and SsnA-JD-R primers every 2 generations, the amplification system is 25 μL: bacterial liquid template 1 μL, 10×PCR Buffer (Mg 2+PLus) 2.5 μL, SsnA-JD-F / R primers 0.5 μL each, 2.5 mM dNTP Mixture 2 μL, 5 U / μL rTaq 0.25 μL, ddH 2 O 18.25 μL. The PCR reaction conditions were as follows: dena...

Embodiment 3

[0081] Determination of Biological Activity of Gene Knockout Mutant SS2-GD01ΔSsnA

[0082] Prepared by Example 1 SsnA The gene knockout mutant strain SS2-GD01ΔSsnA and the wild strain SS2-GD01 were inoculated in TSB medium respectively, cultured with shaking at 37°C to the plateau stage, and the two strains were streaked on the blood plate respectively, and incubated at 37°C, 5% CO 2 After culturing in the incubator for 24 h, observe the colony morphology and the size of the hemolytic ring. Determination of the OD of the two strains in the plateau phase 600 , diluted to OD with TSB medium 600 After =0.2, transfer according to 1% ratio, at 37℃, 5% CO 2 Cultivate in the incubator, take the bacterial solution every 1h to measure the OD 600 , cultivated to 12 h, and draw the growth curve. Such as Figure 11 The SS2-GD01ΔSsnA strain shown had similar growth characteristics to the wild-type strain.

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Abstract

The invention discloses a streptococcussuis serotype 2 SsnA gene knockout mutant strain, and a preparation method and an application of the mutant strain. The mutant strain is formed by substituting an SsnA gene in a streptococcussuis serotype 2 SS2-GD01 strain with a chloramphenicol resistant gene cassette CAT. The mutant strain is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M2014539 on October 31, 2014. The growth characteristic of the SS2-GD01 SsnA strain is similar to a wild strain; the adhesion and invasion capacity of the SS2-GD01 SsnA strain on an Hep-2 cell in vitro is obviously reduced; the virulence of the SS2-GD01 SsnA strain is obviously reduced compared with the wild strain; SsnA participates in a pathopoiesis process of streptococcussuis serotype 2 and is a new virulence correlation factor; and the mutant strain provides an important clue for screening of protective antigens of multivalent subunit vaccine, and can be applied to research and development of streptococcussuis serotype 2 attenuated vaccine.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to type 2 Streptococcus suis SsnA Gene knockout mutant strain and its preparation method and application. Background technique [0002] Streptococcus suis is caused by Streptococcus suis ( Streptococcus suis, S. suis ) is a major bacterial infectious disease that seriously endangers intensive pig production. The disease mainly causes symptoms such as septicemia, meningitis, pneumonia, polyarthritis and serositis in pigs, and the mortality rate of pigs can be as high as 80%, which has caused huge economic losses to the global pig industry. There are many serotypes of Streptococcus suis, among which Streptococcus suis type 2 ( Streptococcus suis serotype 2 , SS2) as an important zoonotic infectious disease pathogen, the most widespread and the most pathogenic, not only affects the healthy development of the breeding industry, but also endangers human health, and can even cause de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74A61K39/09A61P31/04C12R1/46
CPCC07K14/315A61K39/092C12N15/74
Inventor 李淼李春玲宋帅杨冬霞
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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