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

A Streptococcus suis, gene knockout technology, applied in the field of genetic engineering, can solve problems such as no research, and achieve the effects of slowing down the growth rate and enhancing the ability to resist phagocytosis and killing of macrophages

Inactive Publication Date: 2015-01-14
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

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Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1: Construction of gene knockout vector

[0043] (1) According to the upstream and downstream DNA sequences of the htpsC coding gene of the S. suis2 wild strain 05ZYH33 genome, design PCR-specific primers, the base sequence of which is as follows:

[0044] LA1: 5′-CG GAATTC TGAAGCGCAGATAAATC-3' (SEQ ID NO.5, the underline is the introduced EcoR I restriction site)

[0045] LA2: 5′-CG GGATCC AGTCCAAGGTCAAA-3' (SEQ ID NO.6, the underline is the introduced BamH I restriction site)

[0046] RA1: 5′-C GTC GAC TTTTGTCCTCCTAAGCTC-3' (SEQ ID NO.7, the underline is the introduced Sal I restriction site)

[0047] RA2: 5′-CG GCATGC AGGTAGCAGAGATAGTAC-3' (SEQ ID NO.8, the underline is the introduced Sph I restriction site)

[0048] According to the pSET2 plasmid sequence, a pair of specific primers spc-F / spc-R was designed to amplify the entire spc expression cassette with the pSET2 plasmid as a template. The primer sequences are:

[0049] Spc-F: 5'-CC GGATC...

Embodiment 2

[0059] Embodiment 2: Screening and identification of mutant strains

[0060] (1) The gene knockout vector pUC::htpsC was electrotransformed into 05ZYH33 competent

[0061] ①Preparation of S. suis2 wild strain 05ZYH33 competent cells: Pick a single colony of 05ZYH33 and inoculate it with 3ml THB medium, cultivate overnight on a shaking table at 37°C, transfer to THY containing DL-threonine at 1:50 the next day at 37°C Shaker shake culture to OD 600 0.3-0.4, collect the bacteria by centrifugation at low speed at 4°C, wash the bacteria with pre-cooled 10% glycerol 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 Aliquot 50μl / tube and store at -80°C for later use.

[0062] ② Electroporation: Add 10ul of pUC::htpsC plasmid to 50μl of the competent state prepared by the above method, and then add it to the electroporation cup (operated on ice). After electroporation at 22.5kV / cm, 200Ω and 25μF...

Embodiment 3

[0073] Embodiment 3: in vitro experiment

[0074] (1) Gram staining

[0075] According to the instructions of the Gram staining solution produced by Beijing Soleibao Technology Co., Ltd., the wild strain 05ZYH33 and the mutant strain 05ZYH33ΔhtpsC (CGMCC No.7376) were respectively subjected to Gram staining, and it was found that the mutant strain 05ZYH33ΔhtpsC was chain-like The arrangement was more scattered, and the chain length was shorter than that of the wild strain, which indicated that the chain-forming ability of the mutant strain 05ZYH33ΔhtpsC (CGMCC No.7376) was 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 in 3 mL of THB medium containing spectinomycin (100 mg / mL) and without spectinomycin, shaking at 37 °C Incubate overnight. The next day, the overnight cultured bacteria were taken out, the absorbance value at 600nm was...

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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 htpsC gene knockout mutant strain of Streptococcus suis type 2 and an application thereof. Background technique [0002] Streptococcus suis type 2 (S. suis2) is a zoonotic pathogen that endangers the world. The pathogen can not only cause pig meningitis, septicemia, arthritis, pneumonia and endocarditis, but also infect humans, posing a serious threat to the lives 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 swine herds in southern provinces of my country has become increasingly serious, and large-scale outbreaks have occurred from time to time, causing economic losses of billions of yuan each year. In 1998 and 2005, large-scale SS2 infection ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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