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
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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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