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Attenuated enterotoxin C2 superantigen mutant protein, preparation method and application thereof

A mutant protein and superantigen technology, applied in the field of genetic engineering, can solve problems such as limiting clinical application and treatment, achieve high-efficiency expression and purification, reduce toxicity, and reduce toxic and side effects

Inactive Publication Date: 2009-10-21
SHENYANG XIEHE GRP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the function of enterotoxin C2 superantigen must depend on the combination with MHC II molecules in the immune system, this will inevitably lead to its possible combination with MHC II molecules from normal tissues or cells in the human body, thus affecting normal cells. It also produces certain toxic effects; in addition, because Staphylococcus aureus enterotoxin is a kind of bacterial exotoxin, it will produce certain symptoms of toxin syndrome (TSS) and food poisoning on the human body, and it is clinically manifested as vomiting, Diarrhea, and symptoms such as fever, thus limiting its clinical application and therapeutic effect

Method used

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  • Attenuated enterotoxin C2 superantigen mutant protein, preparation method and application thereof
  • Attenuated enterotoxin C2 superantigen mutant protein, preparation method and application thereof
  • Attenuated enterotoxin C2 superantigen mutant protein, preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0033] Having the sequence listing SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 Eight kinds of attenuated superantigen mutant protein gene sequences in the base sequence and sequence listing SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID Eight kinds of attenuated superantigen muteins of NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 amino acid sequences (see sequence listing):

[0034] A superantigen mutein gene having the base sequence of SEQ ID NO: 1 in the sequence table:

[0035] 001 gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt

[0036] 051 tactggtacg atgggtaata tgaaatattt atatgatgat cattatgtat

[0037] 101 cagcaactaa agttatgtct gtagataaat ttttggcaca tgatttaatt

[0038] 151 tataacatta gtgataaaaa actaaaaaat tatgacaaag tgaaaacaga

[0039] 201 gttattaaat gaagatttag caaagaagta caaagatgaa gtagttgatg

[0040] 251 tgtatggatc aaattactat gtaaacgcct atttttcatc caaagataat

[00...

Embodiment 2

[0363] Preparation method of attenuated enterotoxin C2 superantigen mutant protein:

[0364] ① Extraction of Staphylococcus aureus genomic DNA

[0365] Inoculate a single colony of Staphylococcus aureus in 5ml of liquid LB medium, culture overnight on a shaker at 37°C, and collect 1.5ml of the culture by centrifugation to collect the bacteria. Extract Staphylococcus aureus genomic DNA (genome DNA extraction operation presses F. Osper, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Stellar "fine Compilation of Molecular Biology Experiment Guide, New York John Wiley & Sons Publishing House, 1995, third edition, P39-40).

[0366] ②PCR primer design and reaction conditions:

[0367] Synthetic PCR primers were designed to amplify eight superantigen mutant protein gene fragments, using Staphylococcus aureus genomic DNA as a template;

[0368] Using the genomic DNA of Staphylococcus aureus as a template, the primer pair sec2-F: 5'-CGGAATTCGAGAGTCAACCAGA-3' and sec...

Embodiment 3

[0388] Superantigen Activity Detection

[0389] The SPF-grade pure-line mouse Balb / c was sacrificed through the cervical spine, and the spleen was collected under aseptic conditions, crushed lightly, and passed through a 200-mesh sieve. Then the cell suspension passed through the sieve was centrifuged at 1000rpm / min for 5min to collect the cell pellet, the cells were resuspended with 5mL red blood cell lysate, left to stand for 5min and then centrifuged at 1000rpm / min for 5min. Then wash the cells 1-2 times with serum-free 1640 medium (purchased from Gibco), and finally adjust the cell concentration with RPMI-1640 medium containing 10% calf serum (purchased from Gibco) to 8×10 5 cells / well added to 96-well plate. Purified each has sequence listing SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, The eight superantigen mutein gene-encoded proteins with the amino acid sequence in SEQ ID NO: 16 were added to each well at fi...

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Abstract

The invention belongs to the field of genetic engineering, in particular to an attenuated enterotoxin C2 superantigen mutant protein, a preparation method and an application thereof. The attenuated enterotoxin C2 superantigen mutant protein has base sequences in sequence table of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13or SEQ ID NO:15, uses golden staphylococcus DNA as a template, uses a primer for PCR amplification, and then uses sec2-F and sec2-R as primers to obtain a PCR product as a template to carry out the PCR amplification so as to obtain mutant genes which are cloned in pET-28a to construct genetic engineering bacteria of attenuated enterotoxin C2 mutant protein. The mutant protein keeps a certain superantigen activity and obviously decreases the emetic and pyrogenous activity than wild-type attenuated enterotoxin C2 protein, thereby achieving the aim of lessening the toxic side effect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to an attenuated enterotoxin C2 superantigen mutant protein and its preparation method and application. Background technique [0002] Superantigen (SAg) is a group of protein molecules encoded by bacteria or viruses, which have strong immunostimulatory activity on human or other mammalian T lymphocytes at very low concentrations. Unlike traditional antigens, superantigens do not require processing by antigen-presenting cells, and the antigen-binding region on the outside of antigen-presenting cells combines with MHC II (histocompatibility complex) molecules and T cell Vβ regions to form a complex, Thereby a large number of T lymphocytes are stimulated to proliferate, which in turn leads to the release of a large number of cytokines and other effector molecules in vitro or in vivo. Because of the special biological activity and mechanism of action of superantigen, it can be used a...

Claims

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Application Information

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IPC IPC(8): C07K14/31C12N15/31C12N15/70C07K1/22A61K39/085A61P35/00A61P1/08A61P1/12
Inventor 王小刚徐明恺张惠文刘昌孝陈艳陈巨余
Owner SHENYANG XIEHE GRP LTD
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