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Rebuild golden staphylococcus enterotoxin N and preparation and application thereof

A staphylococcus enteric, golden yellow technology, applied in the field of bioengineering, can solve the problems of antigenic determinant and product activity change, poor adsorption selection specificity, etc., and achieve the effect of fast purification and simple steps

Inactive Publication Date: 2007-09-19
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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  • Rebuild golden staphylococcus enterotoxin N and preparation and application thereof
  • Rebuild golden staphylococcus enterotoxin N and preparation and application thereof
  • Rebuild golden staphylococcus enterotoxin N and preparation and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0030] PCR amplification of the mature peptide gene sequence encoding the SEN 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'- gga tcc gaa gtagac aaa aaa ga-3′

[0032] SEQ ID NO.5 (downstream primer, the underlined part is the Xho I restriction site) 5'- ctc gag ata atc ​​atcaat cac tta-3'

[0033] Using the Staphylococcus aureus (FRI 100) genome template, perform PCR amplification according to the following conditions, amplify to obtain a 711bp DNA fragment, see Figure 1 for the gel electrophoresis of PCR results, lane 1 in the figure: nucleic acid standard control (Marker); Swimming lane 2: PCR product of the gene encoding the mature peptide of SEN protein containing restriction sites.

[0034] PCR system:

[0035] h 2 O: 60 μL

[003...

Embodiment 2

[0051] Embodiment 2: the expression of recombinant SEN

[0052] Construction of SEN expression strain: Extract the plasmid from Escherichia coli DH5α containing the pGEX-4T-1-SEN 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 bacteria strain of SEN fusion protein.

[0053] Expression of the fusion protein GST-SEN: 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 SEN

[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 relat...

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Abstract

A recombinant Methicillin-resistant Staphylococcus aureus enterotoxin N belongs to a super antigen with SEQ ID NO.1 amino acid sequence, which is expressed by a recombination of a gene coding SEN from the Staphylococcus aureus and a plasmid vector, and a transformation to the proper host and is obtained a recombinant a highly purified SEN protein by affinity purification. The invention proves the recombinant protein has a typical super antigen activity better than SEC and can be applied in the preparation of activating the lymphocytes multiplication and inhibiting the tumor cell proliferation. The product is suitable for preparing the highly purified enterotoxin with the super antigen activity and exploiting super antigen agent. The invention has a wise design, purifies the target protein with affinity chromatography, has a simple process and a high purification speed.

Description

technical field [0001] The invention belongs to biological engineering, and relates to the preparation of a recombinant staphylococcal enterotoxin N (Staphylococcal Enterotoxin N, SEN) and its application 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), and Staphylococcal Enterotoxin C2 (Staphylococcal Enterotoxin C2). SEC2) etc. [0003] Different from ord...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/31A61K38/16A61K39/085A61P35/00
Inventor 陈枢青潘映秋丁丁李丹曦
Owner ZHEJIANG UNIV
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