Preparation method of exfoliative toxin C (ExhC) proteins of staphylococcus sciuri

A technology of Staphylococcus squirrel and protein is applied in the field of bioengineering to achieve the effect of simple operation and low cost

Inactive Publication Date: 2011-09-07
CHINA AGRI UNIV
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Problems solved by technology

[0007] At present, it has been confirmed that Staphylococcus squirrel has ExhC partial sequence at the gene level (Chen et al., 2007), but whether the bacterium produces desquamation toxin, the pathogenic mechanism of the toxin, and the relationship between the occurrence of exudative dermatitis and desquamation toxin in China are currently It has not been reported yet, so it is necessary to prepare high-purity Staphylococcus squirrel exfoliation toxin C protein. On the one hand, it can be used for basic and clinical research on ExhC. On the other hand, it can be used as an excellent antigen to obtain anti-ExhC monoclonal antibodies through animal immunization. Contribute to the study of the pathogenic mechanism of ExhC

Method used

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  • Preparation method of exfoliative toxin C (ExhC) proteins of staphylococcus sciuri
  • Preparation method of exfoliative toxin C (ExhC) proteins of staphylococcus sciuri
  • Preparation method of exfoliative toxin C (ExhC) proteins of staphylococcus sciuri

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Embodiment 1

[0031] Example 1 Construction of ExhC prokaryotic expression vector

[0032] According to the instructions of the bacterial genomic DNA extraction kit, the genome of Staphylococcus squirrel was extracted. According to the published ExhC gene nucleotide sequence (accession number: AF515455) in GenBank, the primers are designed as follows: upstream primer (ExhC-F) 5'-CCG CCATGG CTATGCATTCAAAACTATTAAGTAAAT-3′, downstream primer (ExhC-R) 5′-ATA GCGGCCGC TTTAATTAATTGTTTGAGATCTCTAATGAG-3′, the upstream and downstream primers respectively introduced NcoI and NotI restriction sites (underlined) and corresponding protective bases. PCR amplification was carried out using the extracted genomic DNA as a template. The reaction program was: 94°C for 4min, 94°C for 30s, 55°C for 30s, 72°C for 1min, cycled 30 times, and the final extension at 72°C for 10min to amplify the full length of ExhC ( figure 1). The PCR products were gel-cut and recovered, digested with NcoI and NotI, and cloned...

Embodiment 2

[0033] Example 2 Expression of recombinant plasmid pET-28a(+)-ExhC in Escherichia coli

[0034] The correctly identified recombinant plasmid pET-28a(+)-ExhC was transformed into E.coli BL 21. Single-picked colonies were inoculated in LB liquid medium (containing 50 μg / ml kanamycin), and cultured overnight at 37°C with shaking. Inoculate in 50ml LB liquid medium according to 1%, shake culture at 37°C until OD 600 0.5-0.7. Add IPTG to a final concentration of 1 mM, collect the cells after induction at 30°C for 6 hours, and set E.coli BL 21 containing only the expression vector pET-28a(+) as a control. The bacterial lysate was separated by 12% SDS-PAGE, stained with Coomassie brilliant blue, and decolorized to obtain a fusion protein of about 32kD in size ( image 3 ). After the proteins were separated by SDS-PAGE, they were electrotransferred to nitrocellulose membrane (NC membrane). Block overnight at 4°C with 5% skim milk powder, incubate with mouse anti-6×His monoclonal ...

Embodiment 3E

[0035] The purification of embodiment 3ExhC protein

[0036] The solubility of the recombinant 6×His fusion protein should be analyzed before protein purification. For the method, refer to the instructions for use of the Bio-Scale Mini IMAC Cartridge. Protein purification is briefly described as follows: Induce 200ml of recombinant bacteria, 8000rpm, centrifuge at 4°C for 10min to collect the bacteria, and use 10ml of engineered bacteria lysate (50mM Tris-HCl, 50mM NaCl, 10% glycerol, 10mM DTT, 100μg / ml lysozyme, pH 8.0) Resuspend the bacterial pellet, inoculate at 4°C for 1 hour, then sonicate the cells in an ice bath, centrifuge at 10,000 rpm, and 4°C for 20 minutes, and the precipitate is the inclusion body. Wash the inclusion bodies with 10ml of engineering bacteria washing solution (50mM Tris-HCl, 50mM NaCl, 10% glycerol, 0.1mMDTT, 1% Triton X-100, pH 8.0), centrifuge at 10000rpm, 4°C for 20min, collect the precipitate, and repeat the washing once. The inclusion bodies w...

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Abstract

The invention relates to a preparation method of exfoliative toxin C (ExhC) proteins of staphylococcus sciuri, which comprises the following steps of: (1) taking staphylococcus sciuri genome DNA as templates, amplifying the overall length of ExhC genes of the staphylococcus sciuri, and cloning the ExhC genes into prokaryotic expression vectors to obtain recombinant plasmids; (2) performing escherichia coli conversion on the recombinant plasmids, and expressing the recombinant plasmids through IPTG (isopropyl thiogalactoside) induced proteins; and (3) extracting the crude extract of the proteins, and purifying ExhC proteins. In the method, the efficient expression of ExhC in escherichia coli is realized successfully through a gene engineering technology, and high-purity and high-concentration ExhC with biological activities is obtained; moreover, the preparation method is simple to operate and low in cost.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to the preparation of staphylococcus squirrel desquamation toxin C protein. Background technique [0002] Staphylococcus sciuri is the pathogen that causes human endometritis, peritonitis, septic shock, urinary tract infection, pelvic inflammatory disease and wound infection. In 2006, our laboratory isolated a strain of highly pathogenic Staphylococcus squirrel from pericardial fluid of pigs with exudative dermatitis (Chen et al., 2007). Porcine exudative dermatitis, also known as "lard skin disease", is a highly contagious acute infectious disease characterized by skin oily exudation, exfoliation, small blister formation, and crusting of the body surface, except Staphylococcus squirrel In addition, the common cause of this disease is some pathogenic Staphylococcus suis (S.hyicus). According to reports, six ecdytic toxins, ExhA, ExhB, ExhC, ExhD, ShetA, and ShetB (Devries...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K14/31C07K1/22C12R1/19
Inventor 郑世军李海花
Owner CHINA AGRI UNIV
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