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

A technology encoding genes and superantigens, applied in the field of genetic engineering, can solve the problems of few structures and functions, unclear research conclusions, etc., and achieve the effects of low serum reactivity, high tumor inhibitory activity, and small 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

[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
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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] Example 2 PCR Amplification of Small Molecule Superantigen Modified Protein Gene

[0162] 1) Extraction of Staphylococcus aureus genomic DNA

[0163] 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 Edited 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 synthesized by Invitrogen Biotechnology Co...

Embodiment 3

[0180] Expression of Transformed Small Molecule Superantigen Protein

[0181] 1) Construction of protein expression vectors encoded by small molecule superantigen modified protein genes: respectively digest the plasmid DNA of the gene cloning vectors pET-28a-sag1, pET-28a-sag2 and pET-28a-sag3 with EcoRI and XhoI, respectively, The gel recovery kit recovers the small molecule superantigen modified protein gene in the sequence table SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. The pET-28a expression vectors that had been digested with the same double restriction enzymes were respectively ligated with T4 DNA ligase to construct expression vectors pET-28a-sag1, pET-28a-sag2 and pET-28a-sag3. E.coli BL21(DE3) competent cells were transformed, and the correct recombinant clones were identified by Sanger dideoxy terminal termination sequencing.

[0182] 2) Induced expression and purification of small-molecule superantigen modified protein: Inoculate a single colony of BL21 (DE3) tr...

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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 Patents(China)
IPC IPC(8): C07K14/31C12R1/19
Inventor 张惠文周隽逸徐明恺陈艳杨宏丽
Owner XIEHE PHARMA FACTORY SHENYANG
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