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Recombinant staphylococcus aureus enterotoxin M and its preparation and application

A technology of staphylococcus enterica and staphylococcus, which is applied in the field of bioengineering, can solve the problems of antigenic determinant and product activity change, poor adsorption selection specificity, etc., and achieves the effect of fast purification and simple steps.

Inactive Publication Date: 2011-05-04
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some people have used genetic engineering methods to prepare Staphylococcus aureus 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. However, the purification steps in these methods All adopt the method of ion exchange, which has the disadvantages of poor adsorption selectivity and poor specificity.
Xu Mingkai et al. cloned the SEC2 gene into pET-28a for expression, but the expressed recombinant product had 36 amino acids more than the natural protein, so the possibility of changing the antigenic determinant and product activity was greater

Method used

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  • Recombinant staphylococcus aureus enterotoxin M and its preparation and application
  • Recombinant staphylococcus aureus enterotoxin M and its preparation and application
  • Recombinant staphylococcus aureus enterotoxin M and its preparation and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the gene cloning of SEM and the construction of pGEM-T-SEM recombinant plasmid

[0030] PCR amplification of the mature peptide gene sequence encoding the SEM protein containing restriction sites: design the following pair of primer sequences:

[0031] SEQ ID NO.4 (upstream primer, the underlined part is the BamH I restriction site): 5'-ca g gat cc t ttt gct att cgc aaa atc ​​ata tcg ca-3′

[0032] SEQ ID NO.5 (downstream primer, the underlined part is the Xho I restriction site) 5'-gc c tcg ag t caa ctt tcg tcc tta taa gat att tct ac-3’

[0033] Using the Staphylococcus aureus (FRI 1230) genome template, perform PCR amplification according to the following conditions, and obtain a 703bp DNA fragment. For the gel electrophoresis diagram of the PCR result, see figure 1 , where Lane 1: Nucleic acid standard control (Marker); Lane 2: PCR product of the mature peptide gene encoding SEM protein containing restriction sites.

[0034] PCR system:

[0035]...

Embodiment 2

[0051] Embodiment 2: the expression of recombinant SEM

[0052] Construction of SEM expression strain: Extract the plasmid from Escherichia coli DH5α containing the pGEX-4T-1-SEM recombinant plasmid, transform it into Escherichia coli BL21 (DE3), and screen positive clones through antibiotic resistance to obtain a large amount of expression GST- Engineering strains of SEM fusion protein.

[0053] Expression of the fusion protein GST-SEM: Inoculate a single colony of the above-mentioned engineered bacteria into 5 mL of LB medium containing ampicillin, culture with shaking at 37° C. for 6 h, and use it as a seed solution. Inoculate the seed solution in 2×YT medium containing ampicillin with an inoculum amount of 1-5%, culture with shaking at 37°C for 4 hours, and add 0.1mol / L isopropyl-β at a volume ratio of 0.01%-0.1% -D-thiogalactoside (IPTG) induced expression for 5h.

Embodiment 3

[0054] Embodiment 3: the purification of recombinant SEM

[0055]Pretreatment of samples: the bacterial solution induced by IPTG was centrifuged at 10,000 rpm at 4°C, and the supernatant was discarded to collect the precipitate. The precipitate was resuspended in phosphate buffered saline (PBS) that was 1 / 10 the volume of the original bacterial solution, and the suspension was placed in a FRENCH cell disruptor and crushed at 700 psi, and the suspension was viscous. Afterwards, continue to sonicate for 2 min with a sonicator to degrade the nucleic acid and reduce the viscosity of the cell lysate. After sonication, 20% Triton-100 was added to the cell lysate to a final concentration of 1%, mixed thoroughly, and left to stand in an ice bath for 30 minutes. After standing still, the cell lysate was centrifuged at 12000 rpm at 4°C for 30 min, and the supernatant was stored at low temperature for later use. The supernatant was sampled for SDS-PAGE detection, and there was a relati...

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Abstract

The recombinant staphylococcus aureus enterotoxin M as one kind of superantigen with the amino acid sequence as shown in SEQ ID No. 1 is obtained through recombining gene originated from staphylococcus aureus and coding SEM with one kind of plasmid vector, transforming to proper host for expressing, and affiliation purifying. It is proved through extracorporeal mouse spleen lymphopoiesis experiment and tumor cell inhibiting experiment that the recombinant protein has typical superantigen activity stronger than that of SEC and may be applied in preparing medicine for stimulating lymphopoiesis and inhibiting tumor cell growth. The present invention is suitable for preparing high purity enterotoxin with superantigen activity and developing superantigen preparation. The present invention has reasonable design, efficient expression on recombinant SEM in the expression strength of about 15-25 % of staphylococcus aureus protein, and other advantages.

Description

technical field [0001] The invention belongs to bioengineering and relates to the preparation of a recombinant Staphylococcal Enterotoxin M (Staphylococcal Enterotoxin M, SEM) and its use in the preparation of drugs for stimulating lymphocyte proliferation and inhibiting tumor cell growth. Background technique [0002] In 1989, the concept of superantigen was proposed by Swedish scientist White. It is a kind of protein produced by bacteria, viruses and parasites that has a powerful stimulating function on lymphocytes. Because its ability to activate T lymphocytes is 2000 times that of ordinary antigens -50000 times, so it is called superantigen. Staphylococcal Enterotoxin (SE) is a superantigen widely studied all over the world. Among them, the most in-depth studies mainly include Staphylococcal Enterotoxin A (SEA), Staphylococcal Enterotoxin B (SEB), Staphylococcal Enterotoxin Type C2 (Staphylococcal Enterotoxin C2, SEC2) etc. [0003] Different from ordinary antigens, e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/31C12N15/31C12N1/21A61K38/16A61P35/00
Inventor 陈枢青潘映秋丁丁
Owner ZHEJIANG UNIV
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