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Gene for encoding HPV (human papilloma virus) 6-type L2N120E7E6 fusion protein, expression vectors, method, bacteria strain and application

A technology of L2N120E7E6 and fusion protein, applied in the field of medical biology, can solve problems affecting the effective presentation of E7 protein and affecting hydrolysis

Active Publication Date: 2013-10-16
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The HPV6 type L2E7 protein is the fusion of the HPV6 type L2 full-length gene and the E7 gene. The L2 full-length protein can only induce weak humoral and cellular immune responses, and the full-length L2 protein (459 amino acids) is fused with the E7 protein. The structure may affect the hydrolysis of the E7 protein (98 amino acids) in the cell, thereby affecting the effective presentation of the T cell epitope of the E7 protein

Method used

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  • Gene for encoding HPV (human papilloma virus) 6-type L2N120E7E6 fusion protein, expression vectors, method, bacteria strain and application
  • Gene for encoding HPV (human papilloma virus) 6-type L2N120E7E6 fusion protein, expression vectors, method, bacteria strain and application
  • Gene for encoding HPV (human papilloma virus) 6-type L2N120E7E6 fusion protein, expression vectors, method, bacteria strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 : Gene sequence designed to express HPV6L2N120E7E6 fusion protein

[0064] According to the amino acid sequence corresponding to the NCBI GenBank HPV6 gene sequence (sequence number NC 001355), the 120 amino acids at the L2 N-terminal of the minor capsid protein sequence of the HPV6 virus were fused with the full-length amino acid sequences of the early protein E7 and E6 proteins, and the design was specifically as follows: The fusion protein shown in SEQ ID NO: 1 (the fusion protein is sequentially L2N120, E7, E6 from the N→C terminal, with a total of 368 amino acids).

[0065]In order to prevent the possibility of homologous sequence gene recombination, the nucleotide sequence of the overlapping part of the E7 protein gene and the E6 protein gene was modified. According to the gene degeneracy, the amino acid sequence of the encoded product protein remained unchanged. Gene 5' end 25bp sequence (this E7 protein gene 5' end 25bp gene overlaps with E6 protein ...

Embodiment 2

[0066] Example 2 : Preparation of L2N120E7E6 target gene fragment by PCR amplification

[0067] In this example, the target gene fragment of L2N120E7E6 was prepared for PCR amplification, and the specific process is described in detail as follows.

[0068] Design primers P1 / P2 (L2N120), P3 / P4 (E7) and P5 / P6 (E6) required for PCR and overlapping PCR according to the relevant nucleotide sequence corresponding to the designed fusion protein for the amplification of the L2N120E7E6 fusion gene . The designed primers are shown in Table 1 in detail. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0069] The DNA screening method of condyloma acuminatum tissue positive for HPV6 gene is as follows: the total DNA was extracted from the condyloma acuminata tissue obtained from gynecological patients in the hospital according to the instructions of the DNA extraction kit (purchased from Beijing Nuokeao Gene Engineering Technology Co., Ltd.). According to the i...

Embodiment 3

[0075] Example 3 : Construction of prokaryotic expression recombinant plasmid pQE30-HPV6L2N120E7E6

[0076] This example is to construct the prokaryotic expression recombinant plasmid pQE30-HPV6L2N120E7E6, which is described in detail as follows.

[0077] The HPV6L2N120E7E6 target gene fragment containing 1107bp amplified by overlapping PCR in Example 2 was digested with a restriction endonuclease. Tao Trading Co., Ltd.) in a water bath at 37°C for 3 hours, and then the fragments were recovered with an agarose gel recovery kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.).

[0078] Then insert the HPV6L2N120E7E6 target gene digested with restriction endonucleases into the Escherichia coli expression plasmid vector pQE30 (product of Qiagen Company, purchased from Beijing Branch of Huamei Bioengineering Company, see figure 1 ) BamHI site, the prokaryotic expression recombinant plasmid pQE30-HPV6L2N120E7E6 with correct insertion was screened by BamHI+HindII...

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Abstract

The invention provides a gene for encoding HPV (human papilloma virus) 6-type L2N120E7E6 fusion protein, expression vectors, a method, a bacteria strain and an application. The invention is characterized in that prokaryotic expression vectors are utilized to fuse HPV 6-type L2N-end 120 amino acids, E7 proteins and E6 proteins, and constructing the encoded gene according to the amino acid of the fusion protein. In the invention, the gene for encoding the HPV 6-type L2N120E7E6 fusion proteins is successfully designed and the expression vectors and the transformation bacteria strain of the high-expression HPV 6-type L2N120E7E6 fusion proteins are successfully constructed; and in the fusion proteins, the immunogenicity is strong, the immunoreaction of specific T cells can be induced, the tumor forming time of mice can be obviously delayed, and the growth of the tumors of 20% mice is completely restrained. The invention can be used for preventing and treating HPV 6-type infection and relevant diseases, such as genital wart.

Description

technical field [0001] The invention belongs to the field of medical biotechnology. Specifically, the present invention relates to a gene expressing human papillomavirus (HPV) type 6 L2N120E7E6 fusion protein, expression vector, method and immunotherapy and prevention application. Background technique [0002] Genital warts (Genital Wart, GW) are one of the most common sexually transmitted diseases in the world. Although GW generally does not lead to death, it can cause psychological shadows in patients and bring about expensive treatment costs. The current treatment methods for GW mainly include: antiproliferative drugs, cryotherapy, destructive therapy, immunomodulators, etc. Incompatibility and recurrence of warts caused by these methods are very common, and the disease has not been cured fundamentally. For effective immunoprevention and treatment of GW methods. [0003] The close relationship between GW and human papillomavirus (Human Papillomavirus, HPV) infection ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N15/70C12N1/21A61K39/12A61K48/00A61P15/00A61P31/20C12R1/19
Inventor 田厚文赵莉任皎庞正冯靖张忠献阮力谭文杰
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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