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Streptococcus suis serotype II htpsC gene knockout mutant strain and application thereof

A gene knockout technology for Streptococcus suis, applied in the field of genetic engineering, can solve problems such as lack of research, and achieve the effect of slowing down the growth rate and enhancing the anti-macrophage phagocytosis and killing ability

Inactive Publication Date: 2013-10-09
中国人民解放军南京军区军事医学研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Reports in recent years have shown that members of the Htp family protein have been found in group A, B, C, and G streptococci, and related studies on the functions of this family member in Streptococcus pyogenes and Streptococcus agalactiae have also been reported, but there is no report yet. Research on the function of the gene in S. suis and its molecular mechanism involved in bacterial pathogenicity

Method used

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  • Streptococcus suis serotype II htpsC gene knockout mutant strain and application thereof
  • Streptococcus suis serotype II htpsC gene knockout mutant strain and application thereof
  • Streptococcus suis serotype II htpsC gene knockout mutant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of gene knockout vector

[0041] (1) According to the upstream and downstream DNA sequences of the htpsC coding gene of the S.suis2 wild strain 05ZYH33 genome,

[0042] Design PCR-specific primers with the following base sequences:

[0043] LA1: 5′-CG GAATTC TGAAGCGCAGATAAATC-3' (SEQ ID NO. 5, the introduced EcoR I restriction site is underlined)

[0044] LA2: 5′-CG GGATCC AGTCCAAGTCAAA-3' (SEQ ID NO. 6, the introduced BamH I restriction site is underlined)

[0045] RA1: 5′-C GTC GAC TTTTGTCCTCCTAAGCTC-3' (SEQ ID NO. 7, the introduced Sal I restriction site is underlined)

[0046] RA2: 5′-CG GCATGC AGGTAGCAGAGATAGTAC-3' (SEQ ID NO. 8, the introduced Sph I restriction site is underlined)

[0047] According to the pSET2 plasmid sequence, a pair of specific primers spc-F / spc-R were designed to amplify the entire spc expression cassette with the pSET2 plasmid as the template. The primer sequences are:

[0048] Spc-F: 5'-CC GGATCC GTTCGT...

Embodiment 2

[0058] Example 2: Screening and identification of variants

[0059] (1) The gene knockout vector pUC::htpsC was electroporated into 05ZYH33 competent cells

[0060] ① Preparation of S.suis2 wild strain 05ZYH33 competent cells: pick a single colony of 05ZYH33 and inoculate it with 3ml of THB medium, shake and culture at 37°C overnight, and transfer to THY containing DL-threonine at 1:50 the next day at 37°C Shaker to OD 600 At about 0.3-0.4, centrifuge at 4°C to harvest the bacteria at a low speed, and wash the bacteria with pre-cooled 10% glycerol for 4 times, each time not less than 25ml, and finally resuspend the bacterial pellet with 0.5ml of 0.3M sucrose containing 15% glycerol, and resuspend the bacteria. Aliquot 50 μl / tube and store at -80°C for later use.

[0061] ②Electrotransformation: Add 10ul of pUC::htpsC plasmid to 50μl of competent cells prepared by the above method and then add it to the electroporation cup (operated on ice). After electroporation at 22.5kV / cm...

Embodiment 3

[0073] Example 3: In vitro experiments

[0074] (1) Gram stain

[0075] According to the instructions of the Gram staining solution produced by Beijing Soleibo Technology Co., Ltd., the wild strain 05ZYH33 and the mutant strain 05ZYH33ΔhtpsC (CGMCC No.7376) were Gram stained respectively. The arrangement is more scattered, and the length of the chain is shorter than that of the wild strain, indicating that the chain-forming ability of the mutant strain 05ZYH33ΔhtpsC (CGMCC No.7376) is weakened.

[0076] (2) Growth characteristics

[0077] Under the same culture conditions, single colonies of 05ZYH33ΔhtpsC (CGMCC No.7376) and wild strain 05ZYH33 were picked and inoculated into 3 mL of THB medium containing spectinomycin (100 mg / mL) and without spectinomycin, respectively, and shaken at 37°C. Incubate overnight. Take out the overnight cultured bacteria the next day, measure the absorbance at 600 nm, and dilute the two with THB medium to about 1 × 10 8 CFU / mL concentration. ...

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Abstract

The invention belongs the field of gene engineering, and relates to a streptococcus suis serotype II htpsC gene knockout mutant strain and an application thereof. The mutant strain 05ZYH33deltahtpsC has the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.7376. A preparation method of the mutant strain comprises the following step of: carrying out gene knockout on an htpsC gene of coded histidine tripolymer protein Htp of a chromosome of a S.suis II field strain 05ZYH33 by adopting a homologous recombinant gene knockout technology so as to obtain the strain, namely the gene knockout mutant strain through verification of combined PCR (polymerase chain reaction) product electrophoresis, RT (reverse transcription)-PCR and DNA (Deoxyribonucleic Acid) sequencing. By using the mutant strain to perform an animal experiment to research pathogenicity of the strain, results show that the toxicity of the mutant strain to the experiment animal is remarkably reduced, and the mutant strain can be applicable to developing a streptococcus suis serotype II attenuated vaccine.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a gene knockout mutant strain of Streptococcus suis type 2 htpsC and its application. Background technique [0002] Streptococcus suis type 2 (S.suis2) is a zoonotic pathogen that threatens the world. The pathogen can not only cause porcine meningitis, sepsis, arthritis, pneumonia and endocarditis, etc., but also infect humans, posing a serious threat to the life safety of relevant practitioners and the general public. According to the antigenicity of its capsular polysaccharide, it can be divided into 35 serotypes, of which type 2 (SS2) is the most virulent and has the highest clinical detection rate. In recent years, the prevalence of S.suis2 in pig herds in southern provinces of my country has become more and more serious, and there have been large-scale outbreaks, causing economic losses of several billion yuan each year. In 1998 and 2005, large-scale SS2 infections broke o...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/09C12N15/63C12N15/66A61K39/09A61P31/04C12R1/46
Inventor 潘秀珍李敏王晶王长军邵珠卿李先富高基民
Owner 中国人民解放军南京军区军事医学研究所
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