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SEC2 mutant gene with increased activity of super-antigen and preparation method thereof

A technology for enhancing activity and mutating genes, which is applied in the field of genetic engineering, can solve problems such as damage, and achieve the effects of convenient purification, high yield, and stable activity

Active Publication Date: 2011-05-11
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since some normal tissues of the human body also express MHC II molecules, the cytotoxicity induced by superantigens usually causes certain damage to such normal cells, so the superantigen can be improved by enhancing the affinity between enterotoxin and MHC II molecules. Antigenic activity is clearly not a viable route

Method used

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  • SEC2 mutant gene with increased activity of super-antigen and preparation method thereof
  • SEC2 mutant gene with increased activity of super-antigen and preparation method thereof
  • SEC2 mutant gene with increased activity of super-antigen and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0031] The SEC2 mutant gene with enhanced superantigen activity has the base sequence in SEQ ID NO: 1 in the sequence table (see sequence table 1). The encoded protein of the mutated gene has the amino acid sequence of SEQ ID NO: 2 in the sequence table (see sequence table 2).

[0032] SEC2 mutant genes with enhanced superantigen activity:

[0033] 001 gagagtcaac cagaccctac gccagatgag ttgcacaaat caagtgagtt

[0034] 051 tactggtttg atggaaaata tgaaatattt atatgatgat cattatgtat

[0035] 101 cagcaactaa agttatgtct gtagataaat ttttggcaca tgatttaatt

[0036] 151 tataacatta gtgataaaaa actaaaaaat tatgacaaag tgaaaacaga

[0037] 201 gttattaaat gaagatttag caaagaagta caaagatgaa gtagttgatg

[0038] 251 tgtatggatc aaattactat gtaaactgct atttttcatc caaagataat

[0039] 301 gtaggtaaag ttacaggtgg taaaacttgt atgtatggag gaataacaaa

[0040] 351 acatgaagga aaccactttg ataatgggaa cttacaaaat gtacttataa

[0041] 401 gagtttatga aaataaaaga aacacaattt cttttgaagt gcaaactgat

[0042] 451 aagaaaagtg taaca...

Embodiment 2

[0076] 1) Construction of pET-28-TM-sec2 plasmid DNA template:

[0077] Design synthetic PCR primers to amplify TM-sec2 sequence by overlapping PCR method

[0078] ①PCR amplification of the left TM sequence

[0079] The PCR reaction system was: 5 μl of 10×Pfu DNA polymerase reaction buffer, 4 uL of dNTP, 20 pmol of upstream and downstream primers, 100 ng of pCVN / Her2 DNA template, 2 U of pfu DNA polymerase, and the volume was made up to 50 μl with sterile ultrapure water.

[0080] The PCR reaction conditions are: the first stage is 95°C, 5 minutes; the second stage is 94°C, 30 seconds; 60°C, 30 seconds; 72°C, 30 seconds; a total of 30 cycles; the third stage is 72°C, 5 minutes.

[0081] The amplification primers were TM-F: 5'-CGGAATTCGCCGAGCAGAGAGC-3', and TM-SEC2-R 5'-TGACTCTCGGATCCCATCGTGTACTTCCG-3'.

[0082] ②PCR amplification of the right sec2 sequence

[0083] The PCR reaction system is: 10×Pfu DNA polymerase reaction buffer 5μl, dNTP 4uL, upstream and downstream prime...

Embodiment 3

[0118] Expression of superantigen mutein SEC2(T20L / G22E)

[0119] 1) Construction of superantigen mutein expression vector: The gene cloning vector pGEM-T-SEC2 (T20L / G22E) plasmid DNA was double-digested with EcoRI and Xho I, and the corresponding digested gene product was recovered with a DNA gel recovery kit. The recovered product was ligated into the pET-28a expression vector that had been cut with the same double restriction enzymes using T4 DNA ligase to construct the expression vector pET-28-SEC2 (T20L / G22E). E.coli BL21(DE3) competent cells were transformed, and the correct recombinant clone was identified by enzyme digestion and DNA sequencing.

[0120] 2) Induced expression and purification of superantigen mutant protein: Inoculate a single colony of BL21 (DE3) transformed with the recombinant expression vector pET-28-SEC2 (T20L / G00E) in liquid LB medium containing 50 μg / ml kanamycin , overnight activation culture, transfer to the next stage with an inoculum size of ...

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Abstract

The invention relates to a gene engineering technology, in particular to an SEC2 mutant gene with increased activity of super-antigen and a preparation method thereof. The SEC2 mutant gene is provided with a base sequence in a sequence table SEQ ID NO:1 and also provided with an amino acid sequence in a sequence table SEQ ID NO:2. The invention establishes the SEC2 mutant gene with remarkably enhanced super-antigen activity and antitumor effect for the first time by using a gene targeted mutagenesis technology and successfully realizes the soluble expression in escherichia coli. The inventioncan provide the gene engineering strain directly used for producing the mutant protein of the super-antigen.

Description

technical field [0001] The invention relates to genetic engineering technology, in particular to a SEC2 mutant gene with enhanced superantigen activity and a preparation method thereof. Background technique [0002] Superantigen (SAg) is a group of protein molecules encoded by bacteria or viruses, which do not require processing by antigen-presenting cells (APCs), but directly bind to MHC II molecules on the cell membrane in the form of complete proteins to form complexes. A very low concentration (1-10ng / ml) can stimulate the proliferation of a large number of T cells with specific Vβ, thereby triggering a strong immune response and producing certain anti-tumor activity. Therefore, as an ideal immunomodulator and potentiator, superantigen is expected to be developed into an effective new antitumor drug for tumor treatment. [0003] Staphylococcal enterotoxins (SEs) are a representative class of bacterial exotoxins. Because of its extremely strong T cell activation functio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C07K14/31C12N15/10C12N15/70A61P35/00A61P37/04
Inventor 王小刚张惠文徐明恺张成刚
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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