SEA mutant gene and use for fusion protein thereof with ScFv gene

A technique for mutating genes and genes, applied in the field of expression and application in Escherichia coli, can solve the problems of lack of selectivity in killing tumor cells and low expression levels, and achieve the effects of small molecular weight, low immunogenicity, and strong penetrability

Inactive Publication Date: 2005-01-26
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The natural SEA coding gene has many rare codons, and its expression level is low when expressed in Escherichia coli; moreover, it lacks selectivity for killing tumor cells, not only can kill tumor cells positive for MHC class II molecules, but also can kill normal organisms. Cells also have a certain killing effect

Method used

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  • SEA mutant gene and use for fusion protein thereof with ScFv gene
  • SEA mutant gene and use for fusion protein thereof with ScFv gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] A mutant gene of sea, having the base sequence shown in the sequence table SEQ ID NO: 1: (wherein the signal peptide gene of SEA is underlined)

[0016] atgaaaaaaa cagcatttac attackttta ttcattgccc taacgttgac aacaagtcca 60

[0017] cttgtaaatg gt agcgagaa aagcgaagaa ataaatgaaa aagatttgcg aaaaaagtct 120

[0018] gaattgcagg gaacagcttt aggcaatctt aaacaaatct atttattacaa tgaaaaagct 180

[0019] aaaactgaaa ataaagagag tcacgatcaa tttttacagc atactatatt gtttaaaggc 240

[0020] ttttttacag atcattcgtg gtataacgat ttattagtag attttgattc aaaggatatt 300

[0021] gttgataaat ataaagggaa aaaagtagac ttgtatggtg cttattatgg ttatcaatgt 360

[0022] gcgggtggta caccaaacaa aacagcttgt atgtatggtg gtgtaacgtt acatgataat 420

[0023] aatcgattga ccgaagagaa aaaagtgccg atcaatttat ggctagacgg taaacaaaat 480

[0024] acagtacctt tggaaacggt taaaacgaat aagaaaaatg taactgttca ggagttggat 540

[0025] cttcaagcaa gacgttattt acaggaaaaa tataatttat ataactctga tgtttttgat 600

[0026] gggaaggttc agaggggatt aatc...

Embodiment 2

[0054] Construction and screening of anti-melanoma single-chain antibody, which has the base sequence shown in the sequence table SEQ ID NO: 2:

[0055] caggtgaagc tgcaggagtc aggaggaggc ttggtgcaac ctggaggatc catgaaactc 60

[0056] tcctgtgttg tctctggatt cactttcagt aattactgga tgaactgggt ccgccagtct 120

[0057] ccagagaagg ggcttgagtg gattgcagaa attagattga aatccaataa ttttgcaaga 180

[0058] tattatgcgg agtctgtgaa agggaggttc accatctcaa gagatgattc caaaagtagt 240

[0059] gtctacctgc aaatgatcaa cctaagagct gaagatactg gcatttatta ctgtaccagt 300

[0060] tatggtaact acgttgggca ctattttgac cactggggcc aagggaccac ggtcaccgtc 360

[0061] tcctcatgtg gaggcggttc aggcggaggt ggctctggcg gtggcggatc ggacattgag 420

[0062] ctcacccagt ctccaaaatt catgtccaca tcagtaggag acagggtcag cgtcacctgc 480

[0063] aaggccagtc agaatgtgga tactaatgta gcctggtatc aacaaaaacc agggcaatct 540

[0064] cctgaaccac tgcttttctc ggcatcctac cgttacactg gagtccctga tcgcttcaca 600

[0065] ggcagtggat ctgggacaga tttcactctc accatcagc...

Embodiment 3

[0102] Construction of superantigen SEA and anti-melanoma ScFv recombinant immunotoxin, which has the base sequence shown in SEQ ID NO: 3 in the sequence table: (wherein the enzyme cleavage junction site is underlined)

[0103] caggtgaagc tgcaggagtc aggaggaggc ttggtgcaac ctggaggatc catgaaactc 60

[0104] tcctgtgttg tctctggatt cactttcagt aattactgga tgaactgggt ccgccagtct 120

[0105] ccagagaagg ggcttgagtg gattgcagaa attagattga aatccaataa ttttgcaaga 180

[0106] tattatgcgg agtctgtgaa agggaggttc accatctcaa gagatgattc caaaagtagt 240

[0107] gtctacctgc aaatgatcaa cctaagagct gaagatactg gcatttatta ctgtaccagt 300

[0108] tatggtaact acgttgggca ctattttgac cactggggcc aagggaccac ggtcaccgtc 360

[0109] tcctcatgtg gaggcggttc aggcggaggt ggctctggcg gtggcggatc ggacattgag 420

[0110] ctcacccagt ctccaaaatt catgtccaca tcagtaggag acagggtcag cgtcacctgc 480

[0111] aaggccagtc agaatgtgga tactaatgta gcctggtatc aacaaaaacc agggcaatct 540

[0112] cctgaaccac tgcttttctc ggcatcctac cgttacactg gag...

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Abstract

The invention relates to a SEA mutant gene and use for fusion protein thereof with ScFv gene, wherein gene engineering means is employed for saltation of super antigen SEA gene, which has the base sequence represented by the sequence table 1, by fusing it with anti-melanoma single-chain antibody (ScFv) gene obtained through bacteriophage displaying technique, a ScFv-SEA recombination antibody is constructed, wherein in vitro cell experiment proves that the fusion protein has inhibitory action to melanin oncocyte superior to other tumor, the fusion protein has evident targeting killing effect to melanin oncocyte.

Description

technical field [0001] The invention belongs to the field of pharmacy, in particular to a mutant gene of the superantigen Staphylococcus aureus enterotoxin A (SEA) and the mutant gene sea m Expression and application of fusion gene with anti-melanoma single-chain antibody (ScFv) in Escherichia coli. Background technique [0002] Superantigen Staphylococcus aureus enterotoxin A (SEA), as an effective immunomodulator and synergist, can achieve the purpose of tumor rejection by activating tumor-specific T lymphocytes. The natural SEA coding gene has many rare codons, and its expression level is low when expressed in Escherichia coli; moreover, it lacks selectivity for killing tumor cells, and it can not only kill tumor cells positive for MHC class II molecules, but also kill normal organisms. Cells also have a certain killing effect. Therefore, it is of great significance to modify the SEA gene [protein] to increase its expression level, endow SEA with targeting, limit the ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61P37/02C07H21/04C12N15/31C12N15/70
Inventor 吴文芳孙静时成波吕安国
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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