Staphylococcus aureus enterotoxin 1 and preparation and use

A staphylococcal intestinal, 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.

Inactive Publication Date: 2007-01-24
HANGZHOU MINSHENG PHARM CO LTD
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  • 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 1 and preparation and use
  • Staphylococcus aureus enterotoxin 1 and preparation and use
  • Staphylococcus aureus enterotoxin 1 and preparation and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: the gene cloning of SEI and the construction of pGEM-T-SEI recombinant plasmid

[0031] PCR amplification of the full-length gene sequence encoding the SEI protein: design the following pair of primer sequences:

[0032] SEQ ID NO.3: (upstream primer) 5'-caa tca taa ctt agt aaa gga aat gcc-3'

[0033] SEQ ID NO.4: (downstream primer) 5'-acg tac aca tta gcc cta gag act t-3'

[0034] Using the Staphylococcus aureus (FRI 100) genome template, carry out PCR amplification according to the following conditions, and obtain a 852bp DNA fragment. For the gel electrophoresis diagram of the PCR result, see figure 1 .

[0035] PCR system:

[0036] h 2 O: 60 μL

[0037] Buffer(10×): 10μL

[0038] Mg 2+ (25mmol / L): 8μL

[0039] BSA (5mg / mL): 10μL

[0040] Primer-up (25μmol / L): 4μL

[0041] Primer-down (25μmol / L): 4μL

[0042] dNTP (20mmol / L): 2μL

[0043] Template (Genome of FRI 100, 10ng / μL): 1μL

[0044] Taq Polymerase (5U / μL): 1μL

[0045] PCR program:...

Embodiment 2

[0070] Embodiment 2: the expression of recombinant SEI

[0071] Construction of SEI expression strain: Extract the plasmid from Escherichia coli DH5α containing the pGEX-4T-1-SEI 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 SEI fusion protein.

[0072] Expression of the fusion protein GST-SEI: 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

[0073] Embodiment 3: the purification of recombinant SEI

[0074] 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 a molecul...

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Abstract

The present invention provides a kind of recombinant staphylococcus aureus enterotoxin I prepared through genetic engineering process, and the protein is one kind of super antigen with the amino acid sequence of SEQ ID No. 1. The recombinant plasmid is reconstructed with pGEX-4T-1 and the nucleotide sequence of SEQ ID No. 2. Extracorporeal experiment shows that the prepared high purity recombinant SEI protein can excite the splenic lymphopoiesis of mouse effectively in dosage depending relationship and the excited splenic lymphocyte of the mouse has obvious tumor cell killing effect. Compared with available staphylococcus aureus enterotoxin C as the main effective component in the staphylococcus aureus filtrate preparation, the recombinant SEI protein has even high super antigen activity and may be prepared into super antigen preparation for inhibiting tumor growth effectively.

Description

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

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/085A61P35/00
Inventor 丁丁李丹曦潘映秋陈枢青
Owner HANGZHOU MINSHENG PHARM CO LTD
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