Staphylococcus aureus enterotoxin E and its preparation and uses

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

Inactive Publication Date: 2007-05-16
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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  • Staphylococcus aureus enterotoxin E and its preparation and uses
  • Staphylococcus aureus enterotoxin E and its preparation and uses
  • Staphylococcus aureus enterotoxin E and its preparation and uses

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0031] SEQ ID NO.3: (upstream primer, the underlined part is the restriction site of BamH I) 5'-atg a gg atc cag cgaaga aat aaa t-3',

[0032] SEQ ID NO.4: (downstream primer, the underlined part is the Xho I restriction site) 5'-atc t ct cga g tc aag ttgtgt ata aat-3',

[0033] The genome template of Staphylococcus aureus (FRI326) was used for PCR amplification according to the following conditions, and a 710bp DNA fragment was amplified. See Figure 1 for the gel electrophoresis of the PCR results.

[0034] PCR system:

[0035] h 2 O: 60 μL

[0036] Buffer(10×): 10μL

[0037] Mg 2+ (25mmol / L): 8μL

[0038] BSA (5mg / mL): 10μL

[0039] Primer-up (25μmol / L): 4μL

[0040] Primer-down (25μmol / L): 4μL

[0041...

Embodiment 2

[0060] Embodiment 2: the expression of recombinant SEE

[0061] Construction of SEE expression strain: Extract the plasmid from Escherichia coli DH5α containing the pGEX-4T-1-SEE 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 SEE fusion protein.

[0062] Expression of the fusion protein GST-SEE: 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. The seed solution was inoculated in 2×YT medium containing ampicillin at an inoculation 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 5 hours.

Embodiment 3

[0063] Embodiment 3: the purification of recombinant SEE

[0064] 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 pellet was resuspended with PBS, which was 1 / 10 of the volume of the original bacterial solution, and the suspension was placed in a FRENCH cell disruptor, 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 relatively dense band at the molec...

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Abstract

The invention discloses a recombination Staphylococcus aureus enteritis toxin E with SEQ ID NO.1 amino acid sequence, which is composed of pGEX-4T-1 and SEQ ID NO.2 nucleotide sequence. The invention utilizes hyperantigen activity to accelerate the breeding of splenocyte and inhibit tumour from growing, which is fit for preparing high-purity enterotoxin and hyperantigen agent.

Description

technical field [0001] The invention belongs to biological engineering, and relates to the preparation of Staphylococcal Enterotoxin E (Staphylococcal Enterotoxin E, SEE) and its use for stimulating lymphocyte proliferation and inhibiting tumor cell growth. Specifically, the gene encoding SEE derived from Staphylococcus aureus is recombined with a plasmid vector, and transformed into a suitable host for expression, high-purity recombinant SEE protein is obtained through affinity purification, and its super Antigen activity is applied to lymphocyte proliferation and tumor cell inhibition, and compared with Staphylococcus aureus enterotoxin C (SEC), which is the main active ingredient of staph aureus filtrate preparation currently used clinically. 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. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31A61K39/085C12N15/09C12N15/31C12N1/21A61P35/00A61P37/04
Inventor 陈枢青张伟潘映秋丁丁
Owner ZHEJIANG UNIV
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