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Attenuated enterotoxin C2 superantigen mutant protein as well as 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

Active Publication Date: 2014-09-24
XIEHE PHARMA FACTORY SHENYANG
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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 as well as preparation method and application thereof
  • Attenuated enterotoxin C2 superantigen mutant protein as well as preparation method and application thereof
  • Attenuated enterotoxin C2 superantigen mutant protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Having a sequence listing 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:13 or SEQ ID NO:15 Eight kinds of attenuated superantigen mutant protein gene sequences in the base sequence and have sequence listing SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 10, SEQ ID NO: Eight kinds of attenuated superantigen mutein bases of ID NO:12, SEQ ID NO:14, SEQ ID NO:16 amino acid sequences (see sequence listing):

[0035] There is the superantigen mutein gene of sequence table SEQ ID NO:1 base sequence:

[0036] 001 gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt

[0037] 051tactggtacg atgggtaata tgaaatattt atatgatgat cattatgtat

[0038] 101 cagcaactaa agttatgtct gtagataaat ttttggcaca tgatttaatt

[0039] 151 tataacatta gtgataaaaa actaaaaaat tatgacaaag tgaaaacaga

[0040] 201gttattaaat gaagatttag caaagaagta caaagatgaa gtagttgatg

[0041] 251tgtatggatc aaattactat gtaaacgcct atttttcatc caaaga...

Embodiment 2

[0364] Preparation method of attenuated enterotoxin C2 superantigen mutant protein:

[0365] ① Extraction of Staphylococcus aureus genomic DNA

[0366] 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 the genomic DNA of Staphylococcus aureus (genome DNA extraction operation according to F. Osper, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Molecular Biology Experiment Guide "New York John Wiley & Sons Publishing House, 1995 third edition P39-40).

[0367] ②PCR primer design and reaction conditions:

[0368] Synthetic PCR primers were designed to amplify eight superantigen mutant protein gene fragments, using Staphylococcus aureus genomic DNA as a template;

[0369] Using the genomic DNA of Staphylococcus aureus as a template, the primer pair sec2-F:5'-CGGAATTCGAGAGTCAACCAGA-3' and sec2(C93A)-R:5'-GATGAAA...

Embodiment 3

[0389] Superantigen Activity Detection

[0390] 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 after passing the sieve was centrifuged at 1000rpm / min for 5min to collect the cell pellet, and 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 1. Eight kinds of superantigen mutein gene coding proteins with amino acid sequence in SEQ ID NO: 16 were added to each well at fina...

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Abstract

The invention belongs to the field of the gene engineering, and concretely relates to a attenuated enterotoxin C2 superantigen mutant protein as well as a preparation method and application thereof. The attenuated enterotoxin C2 superantigen mutant protein has a base sequence in the sequence table SEQ ID No: 7, SEQ ID No: 11, SEQ ID No: 13 or SEQ ID No: 15. A staphylococcus aureus DNA is used as a template, and a primer pair is used for PCR amplification, and then a sec 2-F and a sec 2-R are used as primers, and then an obtained PCR product is used as a template of PCR amplification for obtaining a mutant gene, and the mutant gene is cloned into a pET-28a for constructing a genetically engineered bacterium of the attenuated enterotoxin C2 mutant protein. The mutant protein according to the invention is based on maintenance of a certain superantigen activity, and the emetic and pyrogenicity activities are substantially reduced than those of the wild type enterotoxin C2 protein, thereby achieving the purpose of toxic and side effect reduction.

Description

[0001] This application is a divisional application of the Chinese invention patent application. The filing date of the original application: September 19, 2008, the application number: 200810013284.2, the name of the invention: attenuated enterotoxin C2 superantigen mutant protein and its preparation method and application. Publication number: CN101560248A; During the patent examination process, because the amino acid sequence of the protein involved in the original application has a similar sequence pointed out by the examiner, the applicant hereby submits this divisional application in order to obtain better protection. technical field [0002] 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 [0003] Superantigen (SAg) is a group of protein molecules encoded by bacteria or viruses, which have strong immunostimulatory activity...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/31C12N15/31C12N15/70A61K39/085A61P35/00A61P1/12A61P1/08C12R1/445
Inventor 王小刚徐明恺张惠文刘昌孝陈艳陈巨余
Owner XIEHE PHARMA FACTORY SHENYANG
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