HPV18 type L2NE7E6 fusion protein gene, and expression vector, preparation method, strain and use thereof

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: 2014-06-18
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

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, an

Method used

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  • HPV18 type L2NE7E6 fusion protein gene, and expression vector, preparation method, strain and use thereof
  • HPV18 type L2NE7E6 fusion protein gene, and expression vector, preparation method, strain and use thereof
  • HPV18 type L2NE7E6 fusion protein gene, and expression vector, preparation method, strain and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: Design the optimized codon gene sequence expressed in Escherichia coli of human papillomavirus type 18 L2NE7E6 fusion protein

[0058] First, the HPV18 sequence obtained from cervical cancer patients was compared 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, E6 protein and the stop codon of E7, consider simultaneously that E7, E6 protein is the oncoprotein of HPV18 type, for the safety consideration to vaccine, its relevant amino acid of oncogenic site Mutations were performed by mutating Cys at position 27 of the E7 protein and Glu at position 29 to Gly, 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 sequence as 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]Plasmid pGH-18-33 was synthesized by Beijing Qingke Biotechnology Co., Ltd., which contains the 1-600th nucleotide gene fragment of HPV18L2 protein designed according to the dominant codon of Escherichia coli. The gene fragment, in which the upstream primer P1:L2F contains 6 histidines and Nde I restriction site, the downstream primer P2:L2R contains the BamH I restriction site, the primers are provided by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Synthesized by the company (the specific sequences of the primers are shown in Table 1). Then the amplified PCR fragment was inserted into the cloning vector pMD18-T to construct pMD18-HPV18L2N, and it was confirmed by sequencing that the HPV18L2N gene sequence was consistent with the designed sequence. The specific sequence of HPV18L2N is as follows:

[0062] CGTGCA...

Embodiment 3

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

[0071] This example is to construct the 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 restriction enzymes to obtain

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

[0074] Then the obtained HPV18L2NE7E6 fragment was inserted into the Nde I and BamH I sites of the Escherichia coli expression plasmid pET9a, identified and sequenced by Nde I and BamH I enzyme digestion, and the prokaryotic expression recombina...

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Abstract

The invention provides a human papilloma virus (HPV) 18 type L2NE7E6 fusion protein gene efficiently expressing in Escherichia coli, and an expression vector, a preparation method, a strain and a use thereof L2N protein, E7 protein and E6 protein amino acid sequences of 11th-200th amino acids of an HPV18 type L2 protein are fused, a codon optimization gene suitable for Escherichia coli expression is designed, the codon optimization gene is inserted into a prokaryotic expression vector pET9a to obtain a pET9a-HPV18L2NE7E6 expression vector and its conversion strain, the expression level accounts for 50% of all bacteria, an antibody generated by a pure fusion protein immune mouse can neutralize an HPV18 type pseudovirus in order to generate a specific T cell immune response and obviously postpone the tumor formation time of the mouse, and the growth of 20% of mouse tumors can be completely inhibited. The fusion protein can be used for developing vaccines for preventing and treating HPV18 type infection and relevant 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, plasmid vector, preparation method and its immunoprevention and therapeutic uses. Background technique [0002] Cervical cancer is the second most common female malignant tumor in the world, and it is a serious threat to women's health. High-risk human papillomavirus (Human Papillomavirus, HPV) infection of 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 varies in different regions, and HPV18 ranks second in my country. Infection with HPV18 mainly causes cervical adenocarcinoma. Most of the lesions are located in the internal os of the cervix. It is not easy to ...

Claims

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Application Information

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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 中国疾病预防控制中心病毒病预防控制所
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