Preparation method of recombination staphylococcus aureus enterotoxin C2

A staphylococcal intestinal, golden yellow technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problem of poor adsorption selection specificity, complicated purification steps, low natural toxin production, etc. problem, to achieve the effect of simple steps and fast purification

Inactive Publication Date: 2006-06-14
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low production of natural toxins (mostly less than 100 μg / ml), there are a large number of impurities in the fermentation broth, etc., the later purification steps are complicated, and it is difficult to obtain a large amount of pure products.
Some people have used genetic engineering to prepare staphylococcal enterotoxin superantigens. For example, Jiang Yongqiang et al. cloned SEA, SEB, and SEC1 gene fragments into pBV220, and expressed them in Escherichia coli after heat shock induction. The method of ion exchange has the disadvantage of poor specificity of adsorption selection
Xu Mingkai et al. cloned the SEC2 gene into pET-28a for expression, but the expressed recombinant product had 36 more amino acids than the natural protein, and the possibility of epitope changes and product activity changes was greater.

Method used

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  • Preparation method of recombination staphylococcus aureus enterotoxin C2
  • Preparation method of recombination staphylococcus aureus enterotoxin C2
  • Preparation method of recombination staphylococcus aureus enterotoxin C2

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Construction of expression plasmid pGEX-4T-SEC2 containing recombinant SEC2 gene

[0026]PCR amplification of SEC2 gene sequence: Design the following pair of primer sequences, upstream primer 5'-aca cccaac gta tta gca gag ag-3', downstream primer 5'-ggc aag cac cga agt act tac ct-3'. Using the whole bacterial genome of Shphylococcus aureus FRI 1230 as a template, PCR amplification was performed according to conventional methods, and a 792bp DNA fragment was amplified, as shown in Figure 1.

[0027] Construction of the pGEM-T-SEC2 plasmid containing the recombinant SEC2 gene: the DNA fragment amplified by PCR was cloned into the pGEM-T plasmid, and transformed into Escherichia coli DH5α for amplification. The recombinant plasmid was extracted, Nde I digested and identified, and then sent for sequencing. The result showed that there was a base difference between the sequence and the same protein gene (registration number: AY450554) in the GenBank database: No....

Embodiment 2

[0030] Example 2: Expression of recombinant SEC2

[0031] Construction of recombinant SEC2 expression strain: extract pGEX-4T-SEC2 expression plasmid and transform it into Escherichia coli BL21, and successfully construct an engineering strain that can induce the expression of SEC2.

[0032] Expression of recombinant SEC2: Pick a single colony of the above-mentioned engineered bacteria from the slant and inoculate it in 10 ml of LB medium containing ampicillin, culture it with shaking at 37°C for 6 hours, and use it as a seed solution. The seed solution was inoculated in 2×YT medium containing ampicillin at an inoculum amount of 1-5%, cultured with shaking at 37°C for 4 hours, and 0.1mol / L IPTG was added at a volume ratio of 0.01%-0.1% to induce expression for 4 hours.

Embodiment 3

[0033] Embodiment 3: the purification of recombinant SEC2

[0034] Sample pretreatment: Centrifuge the harvested bacterial liquid at 10000 rpm for 15 min at 4°C, and discard the supernatant. The pellet was resuspended with PBS 10 times the amount of wet bacteria, and placed in a FRENCH cell disruptor, crushed at 700 psi until viscous. Add a small amount of DNase to the homogenate, keep it warm at 4°C for 30-60 minutes, or treat it with an ultrasonic breaker to degrade the nucleic acid and reduce the viscosity of the system. Add 1% volume of 20% Triton X-100, mix well, and place on ice for 30 minutes. The cell lysate was centrifuged at 15,000 rpm for 10 min at 4°C, the supernatant was aspirated, and stored at low temperature until use. Sampling was tested by SDS-PAGE, and there was a very dense band at a molecular weight of about 60,000, which was analyzed by Qauntity One software, and this protein accounted for about 20% of the total bacterial protein, see Figure 6.

[0035...

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Abstract

The invention offers recombination staphylococcus aureus enterotoxin C2(SEC2) making method. It can realize by the following steps: amplifying and preparing SEC2 gene; connecting SEC2 gene and pronucleus expression plasmid; transforming recombination plasmid to expression host to efficiently express; purifying recombination SEC2. The method efficiently expresses recombination SEC2; and it is solubility, and has no occlusion body. Its advantages are simple steps, and quick purifying speed. Compared with corresponding nature albumen, the gained recombination has the same super antigen and immunology activity.

Description

technical field [0001] The invention relates to a method for preparing recombinant Staphylococcus aureus enterotoxin C2 (SEC2) by means of genetic engineering, specifically, it involves recombining the SEC2 gene derived from Staphylococcus aureus with a plasmid vector and transforming it into A method for expressing, isolating and purifying recombinant SEC2 in an expression host. Background technique [0002] Superantigen (Superantigen, SAg) is a group of protein molecules encoded by bacteria or viruses, which can directly bind to the outside of the MHCII molecular antigen-binding groove on the APC membrane in the form of complete protein molecules without being processed by antigen-presenting cells (APC); And superantigens combine with most allelic products of MHC class II molecules, unlike common antigens that only bind with limited specific MHC class II allelic products, resulting in specific V β Segmental T cells are activated and proliferated in large numbers, and the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K14/31C12N1/21C12N15/31C12N15/70C12P21/02
Inventor 陈枢青应跃斌薛乔李丹曦
Owner ZHEJIANG UNIV
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