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HPV18 type l2ne7e6 fusion protein gene, expression vector, method, bacterial strain and application

A 18L2NE7E6, L2NE7E6 technology, applied in the field of human papillomavirus type 18 L2NE7E6 fusion protein, can solve the problem of less research

Active Publication Date: 2018-01-23
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

However, there are relatively few studies on therapeutic vaccines for HPV18. Only the HPV18E6 protein using DNA as a carrier can induce specific cellular immune responses in mice, and the HPV18E6E7 fusion protein expressed by vaccinia virus as a carrier can induce post-immunization. The human body produces different degrees of specific cellular immune responses

Method used

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  • HPV18 type l2ne7e6 fusion protein gene, expression vector, method, bacterial strain and application
  • HPV18 type l2ne7e6 fusion protein gene, expression vector, method, bacterial strain and application
  • HPV18 type l2ne7e6 fusion protein gene, expression vector, method, bacterial strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Designing the optimized codon gene sequence of human papillomavirus type 18 L2NE7E6 fusion protein expressed in E. coli

[0058] First, the HPV18 sequence isolated and sequenced from cervical cancer patients was aligned with the sequence in Genebank, which was consistent with AY262282, and then the nucleotide sequence of the minor capsid protein L2N protein was fused with the early protein E7 and E6 nucleotide sequences. And remove the start codon of E7 and E6 proteins and the stop codon of E7, and consider that E7 and E6 proteins are HPV18 oncoproteins. For the safety of the vaccine, the amino acids related to the oncogenic site are taken into account. Mutation was performed by mutating Cys at position 27 and Glu at position 29 of E7 protein to Gly, and the amino acid at position 65 of E6 was mutated from Cys to Gly, and the amino acid sequence encoding the HPV18L2NE7E6 fusion protein was designed to obtain the specific SEQ ID NO. : the fusion protein shown ...

Embodiment 2

[0060] Example 2: Synthesis of codon-optimized gene sequences and construction of cloning plasmids containing 18L2NE7E6 target gene fragments

[0061]The plasmid pGH-18-33 was synthesized by Beijing Qingke Biotechnology Co., Ltd., which contains the nucleotide gene fragment of HPV18L2 protein from positions 1 to 600 designed according to the dominant codon of Escherichia coli, and the L2N protein was first increased by PCR method. Gene fragment, the upstream primer P1: L2F contains 6 histidines and Nde I restriction site, and the downstream primer P2: L2R contains BamH I restriction site, the primers are provided by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Synthesized by the company (the specific sequences of primers are shown in Table 1). Then the amplified PCR fragment was inserted into the cloning vector pMD18-T to construct pMD18-HPV18L2N. The sequence of the HPV18L2N gene was confirmed to be consistent with the designed sequence by sequencing. The spec...

Embodiment 3

[0070] Example 3: Construction of prokaryotic expression recombinant plasmid pET9a-HPV18L2NE7E6

[0071] This example is to construct a prokaryotic expression recombinant plasmid pET9a-HPV18L2NE7E6, which is described in detail as follows.

[0072] The pMD18T-HPV18L2NE7E6 cloning plasmid constructed in Example 2 was digested with enzymes to obtain

[0073] The HPV18L2NE7E6 gene fragment, the specific operation of the enzyme digestion and digestion is as follows: first, digest with Nde I enzyme at 37°C for 2 hours, then recover with agarose gel recovery kit, and then digest with BamH I enzyme at 37°C for 2 hours, and finally use The HPV18L2NE7E6 fragment was recovered by agarose gel recovery kit.

[0074] Then the obtained HPV18L2NE7E6 fragment was inserted into the Nde I and BamH I sites of the E. coli expression plasmid pET9a, identified and sequenced by Nde I and BamH I digestion, and screened to obtain the correct inserted prokaryotic expression recombinant plasmid pET9a-H...

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Abstract

The invention provides a human papillomavirus (HPV) type 18 L2NE7E6 fusion protein gene, expression vector, method, bacterial strain and application highly expressed in Escherichia coli. In the present invention, the amino acid sequences of L2N protein, E7 protein and E6 protein at the 11-200th amino acid position of HPV18 L2 protein are fused, and a codon-optimized gene suitable for expression in Escherichia coli is designed, which is inserted into the prokaryotic expression vector pET9a to obtain pET9a- The expression level of HPV18L2NE7E6 expression vector and its transformed strains accounted for 50% of the whole bacteria. The purified fusion protein was immunized to mice, and the antibodies produced could neutralize HPV18 pseudoviruses, generate specific T cell immune responses, and significantly delay small At the time of mouse tumor formation, 20% of the mouse tumor growth was completely inhibited. The invention can be used for the research and development of vaccines for preventing and treating HPV18 infection and related diseases.

Description

technical field [0001] The invention belongs to the field of medical biotechnology. Specifically, the present invention relates to a human papillomavirus (HPV) type 18 L2NE7E6 fusion protein, its encoding gene, a plasmid vector, a preparation method, and its immunoprophylactic and therapeutic uses. Background technique [0002] Cervical cancer is the second most common female malignant tumor in the world, which is a serious threat to women's health. High-risk human papillomavirus (HPV) infection in the reproductive tract is closely related to the occurrence of cervical cancer. More than 90% of cervical cancer patients can detect HPV-DNA in cervical samples, of which HPV16 has the highest detection rate, and other The detection rate of high-risk types is different in different regions, and the second most important type in my country is HPV18. Infection with HPV18 mainly causes cervical adenocarcinoma, and its lesions are mostly located in the internal cervix, which is not e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21A61K48/00A61K39/295A61K39/12A61P31/20A61P35/00C12R1/19
Inventor 田厚文赵莉任皎冯靖庞正阮力谭文杰
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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