Small molecular superantigen modified protein, its encoding gene, preparation process and application thereof

A superantigen and small molecule technology, applied in the field of genetic engineering, can solve the problems of unclear research conclusions, few structures and functions, and achieve the effects of high tumor inhibitory activity, low serum reactivity, high expression and purification

Active Publication Date: 2012-10-10
XIEHE PHARMA FACTORY SHENYANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Enterotoxin C2 (SEC2) is a member of the Staphylococcus aureus enterotoxin family. There are not many studies on its structure and function at home and abroad, and the conclusions of the existing studies are not yet clear.

Method used

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  • Small molecular superantigen modified protein, its encoding gene, preparation process and application thereof
  • Small molecular superantigen modified protein, its encoding gene, preparation process and application thereof
  • Small molecular superantigen modified protein, its encoding gene, preparation process and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1) Small molecular superantigen modified body protein gene having the base sequences of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 in the sequence table:

[0032] Small molecule superantigen modified body protein gene with sequence table SEQ ID NO: 1 base sequence:

[0033] 001 tcaagtgagt ttactggtac gatgggtaat atgaaatatt tatatgatga

[0034] 051 tcattatgta tcagcaacta aagttatgtc tgtagataaa tttttggcac

[0035] 101 atgatttaat ttataacatt agtgataaaa aactaaaaaa ttatgacaaa

[0036] 151 gtgaaaacag agtttattaaa tgaagatta gcaaagaagt acaaagatga

[0037] 201 agtagttgat gtgtatggat caaattacta tgtaaactgc tatttttcat

[0038] 251 ccaaagataa tgtaggtaaa gttacaggtg gtaaaacttg tatgtatgga

[0039] 301 ggaataacaa aacatgaagg aaaccacttt gataatggga acttataa

[0040] Information on SEQ ID NO.1 (see Sequence Listing)

[0041] (a) Sequence features:

[0042] * Length: 348 bp

[0043] *Type: nucleic acid

[0044] * Chain type: double chain

[0045] *Topology: Linear

[0046] (b) Molecular ty...

Embodiment 2

[0161] Embodiment 2 PCR amplification of small molecule superantigen recombinant protein gene

[0162] 1) Extraction of Staphylococcus aureus genomic DNA

[0163] A single colony of Staphylococcus aureus was inoculated into 5 ml of liquid LB medium, cultured on a shaker at 37°C overnight, and 1.5 ml of the culture was collected by centrifugation. Extraction of Staphylococcus aureus genomic DNA (the genomic DNA extraction procedure was performed according to F. Osber, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Stall "Essential" Compilation of Molecular Biology Experiment Guide, New York John Wiley & Sons Publishing House, 1995 Third Edition P39-40).

[0164] 2) PCR primer design and reaction conditions:

[0165] Use the primer design software Primer5.0 to design the end primers shown below and the mutation primers shown in Table 1:

[0166] F2: 5'-CGGAATTCGAGAGTCAACCAGA-3'

[0167] R2: 5'-TCGCTCGAGTTATCCATCTTTGTTG-3'

[0168] The primers used were sy...

Embodiment 3

[0180] Expression of Small Molecule Superantigen Modified Protein

[0181] 1) Construction of the protein expression vector encoding the protein gene of the small molecule superantigen transformant: The gene cloning vectors pET-28a-sag1, pET-28a-sag2 and pET-28a-sag3 plasmid DNA were digested with EcoRI and XhoI, respectively. The gel recovery kit recovers the small molecule superantigen recombinant protein genes in the sequence listing SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. T4 DNA ligase was used to ligate into the pET-28a expression vector digested by the same double enzyme, and the expression vectors pET-28a-sag 1, pET-28a-sag2 and pET-28a-sag3 were constructed. E. coli BL21 (DE3) competent cells were transformed, and the correct recombinant clones were identified by Sanger dideoxy end termination sequencing.

[0182] 2) Induction, expression and purification of small molecule superantigen recombinant protein: inoculate a single colony of BL21(DE3) transformed with t...

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PUM

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Abstract

The present invention relates to a gene engineering technology, in particular to a small molecular superantigen modified protein, its encoding gene, a preparation process and an application thereof. The invention provides a small molecular superantigen modified protein, its encoding gene, a preparation process and an application thereof. The small molecular superantigen modified protein has an amino acid sequence of sequence table SEQIDNO:2, SEQIDNO:4 or SEQIDNO:6. The encoding gene of the small molecular superantigen modified protein has a base sequence of sequence table SEQIDNO:1, SEQIDNO:3 or SEQIDNO:5.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a small-molecule superantigen modified protein and its preparation method and application. Background technique [0002] Superantigen (SAg) is a group of protein molecules encoded by bacteria or viruses, which stimulates the proliferation of most T cells at very low concentrations (1-10 ng / mL), and has a super-strong function of enhancing the body's immune response and certain anti-inflammatory effects. tumor activity. Therefore, superantigen is an excellent immunomodulator and synergist, and it is expected to be developed into a potential new antitumor drug for tumor treatment. [0003] Staphylococcal enterotoxins (SEs) are a representative class of microbial exotoxins. Because of its extremely strong T cell activation function, it has become a typical microbial superantigen and has been widely valued by people. In recent years, people have done a lot of research work on th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31C12N15/31C12N15/70A61K38/16A61P37/02A61P35/00C12R1/19
Inventor 张惠文周隽逸徐明恺陈艳杨宏丽
Owner XIEHE PHARMA FACTORY SHENYANG
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