Human papilloma virus (HPV) fusion protein, gene, carrier, strain, preparation method and application

A fusion protein and strain technology, applied in the field of bioengineering, can solve the problems that virus particles are unlikely to develop into vaccines, HPV is difficult to cultivate, etc.

Inactive Publication Date: 2010-06-16
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the difficulty of culturing HPV in vitro and its carcinogenicity, complete virus particles are unlikely to be developed into vaccines, and only genetically engineered vaccines can be developed

Method used

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  • Human papilloma virus (HPV) fusion protein, gene, carrier, strain, preparation method and application
  • Human papilloma virus (HPV) fusion protein, gene, carrier, strain, preparation method and application
  • Human papilloma virus (HPV) fusion protein, gene, carrier, strain, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Design and synthesis of human papillomavirus type 16 L2E7 fusion protein Escherichia coli expression optimization codon gene

[0052] The HPV16 late gene L2 and early gene E7 isolated and cloned from cervical cancer patient specimens were sequenced, and according to the obtained HPV16 late gene L2 and early gene E7 encoded polypeptide amino acid sequences, the E7 protein was combined with the two binding sites of pRB The codons of the key amino acids cysteine ​​at position 24 and glutamic acid at position 26 were mutated to glycine codons GGT and GGG, respectively, to eliminate their tumor transformation activity. The minor capsid protein sequence L2 of the HPV16 virus is combined with the E7 protein sequence of the HPV16 virus to be designed as a fusion protein as follows:

[0053] MRHKRSAKRTKRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQ

[0054] ILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLGTRPPTATDTLAPV

[0055] RPPLTVDPVGPSDPSIVSLVEETSFIDAGAPTSVPSPSIPPDVSGFSIT

[0056] TSTD...

Embodiment 2

[0109] The above sL2E7 nucleotide sequence was sent to Gene Synthesis Company for synthesis, and the company cloned it into pUC18 to obtain the recombinant cloning plasmid pUC18sL2E7 of the synthetic gene. For the structure of the recombinant cloning plasmid, see figure 1 .

[0110] The pUC18 used in this example is a commercially available plasmid vector, and its specific structure can be found in the literature: Sambrook J, Fritsch EF, and Manny Artis T, Molecular Cloning Experiment Guide. 1992. Second Edition (Beijing): Science Press: Page 9.

Embodiment 3

[0112] The synthesized and cloned L2E7 optimized gene was digested by enzyme digestion. The operation of the enzyme digestion and digestion was as follows: first digested with Nde I enzyme in a 37°C water bath for 2 hours, then recovered with an agarose gel recovery kit, and then BamH I enzyme 37 Digest in water bath at ℃ for 2 hours, and finally recover the fragments with an agarose gel recovery kit. The Nde I and BamH I enzymes used are Biolabs products and purchased from Beijing North Yitao Trading Co., Ltd.; Agarose gel recovery kit It is a product of Beijing Tiangen Biochemical Technology Co., Ltd., and then inserted into the Nde I and BamH I sites of the Escherichia coli expression vector pET9a. The prokaryotic expression recombinant plasmid pET9asL2E7 with correct insertion was obtained after Nde I and BamH I enzyme digestion and sequencing screening , its structure diagram is as follows figure 2 shown. The pET9a used is a commercially available expression plasmid vec...

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Abstract

The invention relates to a codon-optimized gene of a 16-type L2E7 recombinant protein of human papilloma virus (HPV), which can be expressed efficiently in colibacillus. The optimized gene can be expressed efficiently by being inserted in a prokaryotic expression vector pET9a, and the expression level accounts for 50% of all viruses; an expression protein is immune to mice after being purified. Proved by experiment results, the protein has the same immunogenicity with a protein which is not expressed by the optimized gene and can induce the release of specific gamma-1NF; proved by tumor growth inhibition animal experiments, protein immunity has evident inhibition effect on tumor growth and the growth of 90% of mouse tumor cells can be inhibited completely. The prokaryotic high-efficiency expression system of L2E7 recombinant protein, which is established by the optimized gene, can be used in the pilot scale production and drug discovery of preventive and curative vaccines of HPV16 infection and relevant cervical carcinoma.

Description

technical field [0001] The invention belongs to the technical field of bioengineering. Specifically, the present invention relates to a human papillomavirus (HPV) type 16 L2E7 fusion protein, its encoding gene, plasmid vector, expression strain, preparation method and its immunoprevention and therapeutic uses. Background technique [0002] Statistics from the World Health Organization show that cervical cancer ranks second in cancer mortality among women worldwide, and even ranks first in some developing countries. There are approximately 500,000 new cases of cervical cancer and approximately 200,000 deaths from cervical cancer each year [1-3] , 80 percent of which are in developing countries. my country is one of the countries with a high incidence of cervical cancer. According to incomplete statistics, the annual incidence of cervical cancer in my country is about 138,000, and about 50,000 patients die of cervical cancer every year. Existing research data have proved th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/025C12N15/37C12N15/70C12N1/21C12P21/04A61K39/12A61P31/20A61P35/00C12R1/19
Inventor 田厚文高见赵莉任皎张卉阮力
Owner 中国疾病预防控制中心病毒病预防控制所
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