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Prepn and application of recombinant staphylococcus aureus enterotoxin O

A staphylococcus entero, golden yellow technology, applied in the field of bioengineering, can solve the problems of poor adsorption selection specificity, changes in antigenic determinants and product activity, etc., and achieves the effect of simple steps and fast purification.

Active Publication Date: 2007-10-03
HANGZHOU ENSHI GENE TECH DEV
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  • Abstract
  • Description
  • Claims
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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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  • Prepn and application of recombinant staphylococcus aureus enterotoxin O
  • Prepn and application of recombinant staphylococcus aureus enterotoxin O
  • Prepn and application of recombinant staphylococcus aureus enterotoxin O

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the gene cloning of SEO and the construction of pGEM-T-SEO recombinant plasmid

[0029] PCR amplification of the mature peptide gene sequence encoding the SEO protein containing restriction sites: design the following pair of primer sequences:

[0030] SEQ ID NO.3 (upstream primer, the underlined part is the restriction site of BamH I): 5'- gga tcc tcc aatgaa gaa gat c-3′

[0031] SEQ ID NO.4 (downstream primer, the underlined part is the Xho I restriction site) 5'- ctc gag ttt cag attgtt atg-3'

[0032] Using the Staphylococcus aureus (FRI 100) genome template, perform PCR amplification according to the following conditions, and obtain a 721bp DNA fragment. See Figure 1 for the gel electrophoresis of PCR results, where lane 1: nucleic acid marker; lane 2: enzyme-containing The PCR product of the mature peptide gene encoding the SEO protein at the cleavage site. .

[0033] PCR system:

[0034] h 2 O: 60 μL

[0035] Buffer(10×): 10μL

[0036] Mg...

Embodiment 2

[0050] Embodiment 2: Expression of recombinant SEO

[0051] Construction of SEO expression strain: extract the plasmid from Escherichia coli DH5α containing the pGEX-4T-1-SEO 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 SEO fusion protein.

[0052] Expression of the fusion protein GST-SEO: 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

[0053] Embodiment 3: the purification of recombinant SEO

[0054] 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 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) d...

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Abstract

The present invention provides process of preparing recombinant staphylococcus aureus enterotoxin O, as one kind of superantigen with the amino acid sequence as shown in SEQ ID No. 1. The present invention constructs one kind of recombinant plasmid for expression staphylococcus aureus enterotoxin O via recombining pGEX-4T-1 and the polynucleotide sequence of SEQ ID No. 1, and obtains initial fusion GST-SEO protein accounting for 15-25 % of total bacterial protein content. The present invention utilizes the recombinant staphylococcus aureus enterotoxin O with superantigen activity in promoting extracorporeal spleen lymphopoiesis and inhibiting extracorporeal tumor cell growth. It is proved that the recombinant staphylococcus aureus enterotoxin O has superantigen activity similar to that of SEC. The present invention is suitable for use in preparing high purity enterotoxin with superantigen activity and developing superantigen preparation.

Description

technical field [0001] The invention belongs to bioengineering, and relates to the preparation of a recombinant Staphylococcal Enterotoxin O (Staphylococcal Enterotoxin O, SEO) and its use for stimulating lymphocyte proliferation and inhibiting tumor cell growth. Specifically, the gene encoding SEO derived from Staphylococcus aureus is recombined with a plasmid vector, and transformed into a suitable host for expression, high-purity recombinant SEO 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 lymphoc...

Claims

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Application Information

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IPC IPC(8): C12N15/09C07K14/31C12P21/02A61K39/085A61P35/00C12N15/31C12N1/21
Inventor 陈枢青潘映秋丁丁
Owner HANGZHOU ENSHI GENE TECH DEV
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