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Modified HPV E6-E7 fusion gene and coding protein thereof

A HPV16E6-E7, fusion gene technology, applied in the field of fusion genes and their encoded proteins

Inactive Publication Date: 2010-05-26
曾毅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In addition, since the E6 and E7 genes of HPV16 are both oncogenes, the fusion genes of the two must have potential carcinogenicity.

Method used

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  • Modified HPV E6-E7 fusion gene and coding protein thereof
  • Modified HPV E6-E7 fusion gene and coding protein thereof
  • Modified HPV E6-E7 fusion gene and coding protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Codon optimization of HPV16 E6E7

[0041] In order to increase the expression of the HPV16 E6E7 gene in mammalian cells, especially human cells, codon optimization was performed on the HPV16 E6E7 gene in this embodiment.

[0042] According to the partial tropism of human gene codons, a codon-optimized HPV16 E6E7 fusion gene was designed by using the most frequently used codons and the second most frequently used codons in human genes, named ofE6E7, and its encoded amino acid sequence contains the complete wild-type The amino acid sequences of HPV16 E6 protein (Genebank ACCESSION: AAA46939) and E7 protein (Genebank ACCESSION: AAA46940) are directly fused with the amino acid sequence of E6 protein at the amino terminal and the amino acid sequence of E7 protein at the carboxyl terminal, wherein the amino acid sequence of E6 protein is The 136th arginine and the 32nd seramino in the amino acid sequence of the E7 protein use the codons AGG and TCC, which are the second most ...

Embodiment 2

[0045] Synthetic codon-optimized HPV16 E6E7 fusion gene

[0046] In this example, the ofE6E7 gene described in Example 1 was spliced ​​and synthesized by overlapping extension PCR (overlap extension PCR, OE PCR).

[0047] Primer design

[0048]Referring to the codon-optimized nucleotide sequence ofE6E7, 10 long-chain primers (A1, A2, B1, B2, C1, C2, D1, D2) with a length of about 89 oligonucleotides containing overlapping sequences were designed and synthesized. ), and 10 short-chain primers (p1, p2, p2, p4, p5, q1, q2, q3, q4, q5) that are used to connect these long-chain primers with a length of about 25 oligonucleotides, of which Two short-chain primers (p1 and q10) contain restriction enzymes Kpn I and Xba I cutting sites. The primers were synthesized by entrusting Beijing Qingke Biotechnology Co., Ltd., soluble in ddH 2 In O, the concentration of long-chain primers was 10 μmol, and the concentration of short-chain primers was 25 μmol. The primer sequences are shown i...

Embodiment 3

[0060] Embodiment 3: Site-directed mutagenesis of ofE6E7 gene

[0061] In this embodiment, site-directed mutagenesis is performed on the obtained optimized fusion HPV16 E6E7 gene ofE6E7 gene to prepare a codon-optimized mutant HPV16 E6E7 fusion gene to eliminate the carcinogenicity of the HPV16 E6E7 gene to eliminate the carcinogenicity of the HPV16 E6-E7 fusion gene .

[0062] Specifically, in the amino acid sequence part of the E6 protein: 57Leu(CTG)→Gly(GGC) and 113Cys(TGC)→Arg(AGA), in the amino acid sequence part of the E7 protein: 24Cys(TGC)→Gly(GGC), 26Glu (GAG)→Gly(GGC), 58Cys(TGC)→Gly(GGC) and 91Cys(TGC)→Gly(GGC). The corresponding residue positions of L57G, C113R of the above-mentioned E6 protein and C24G, E26G, C58G, and C91G of the E7 protein in the amino acid sequence of the fusion protein (E6 is at the 5' end, and E7 is at the 3' end) are: L57G, C113R, C182G, E184G, C216G, and C249G. In this example, three kinds of codon-optimized fusion genes after site-direc...

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Abstract

A code optimizing HPV16 E6-E7 fused gene, fused gene with carcinogenicity elimination by fixed-point mutagen and it encoded fused protein are disclosed. The modified fused genes are increased in mammal cell expression. It has excellent cell conversion activity and biological safety. It can be used to offer excellent provisional antigen gene for constructing related DNA vaccine.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and tumor treatment. Specifically, the present invention relates to a codon-optimized HPV16E6E7 fusion gene and its encoded protein, as well as three potential carcinogenicity-eliminated fusion genes obtained by site-directed mutagenesis of the codon-optimized HPV16 E6E7 fusion gene and its encoded protein. coded protein. Background technique [0002] Human papillomavirus (Human Papillomavires, HPV) is a kind of DNA virus with strict host range and tissue specificity. It mainly infects human skin or mucosal epithelial cells, causing benign and malignant lesions at the infection site. With the deepening of HPV research, it is found that about 80% of cervical cancers are related to four types of HPV infection, namely HPV16, 18, 31 and 45, and 50% of cervical cancers are related to HPV16 infection (MH Stoler . Int J Gynecol Patho, 2000, 19(1): 16-28). According to statistics, there are about 51...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C07K19/00A61K48/00A61P35/00
Inventor 曾毅谢强周玲李泽林盛望
Owner 曾毅
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