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Gene, expression vector, expression method, expression cell and application of human papilloma virus (HPV) 16 E7E6 fusion protein

A fusion protein and expression carrier technology, applied in the field of medical biology, can solve problems affecting the effect of immunotherapy and achieve strong immunogenic effects

Active Publication Date: 2011-04-06
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

[0005] Adenovirus vector vaccine has many advantages, but there are also shortcomings. The neutralizing antibody produced by its original high background infection affects its wide use in new vaccines. In addition, the antiviral antibody produced after immunization may inhibit re-infection Immunity, thus affecting the effect of immunotherapy, but people have overcome this shortcoming through the treatment of heterologous vaccine combined immunization

Method used

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  • Gene, expression vector, expression method, expression cell and application of human papilloma virus (HPV) 16 E7E6 fusion protein
  • Gene, expression vector, expression method, expression cell and application of human papilloma virus (HPV) 16 E7E6 fusion protein
  • Gene, expression vector, expression method, expression cell and application of human papilloma virus (HPV) 16 E7E6 fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 : Design and synthesis of human papillomavirus type 16 E7E6 fusion protein and codon-optimized fusion gene suitable for expression in mammalian cells

[0048] In this example, the human papillomavirus type 16 E7E6 fusion protein was designed and synthesized, and a codon-optimized fusion gene suitable for expression in mammalian cells was designed. The specific process is as follows.

[0049] According to the gene coding region sequence of HPV16 type E7, E6 (Genebank No. NC_001526), ​​the amino acid sequence encoded by HPV16 type E7, E6 gene was obtained, and the two key amino acids of the binding site of E7 protein and pRB were cysteine ​​at position 24 and cysteine ​​at position 24. Glutamic acid at position 26 is changed to glycine, and leucine at position 57, a key amino acid in the P53 degradation active site of E6 protein, is changed to glycine to eliminate its tumor transformation activity. Then connect the C-terminal of the amino acid sequence of the ...

Embodiment 2

[0051] Example 2 : Construction of cloning plasmid pUC18-HPV16smE7E6

[0052] This example is to construct the cloning plasmid pUC18-HPV16smE7E6.

[0053] The nucleotide sequence of the HPV16SmE7E6 fusion gene designed in Example 1 was sent to Beijing Qingke Biotechnology Co., Ltd. for synthesis, and the company cloned it into pUC18 to obtain the cloning plasmid pUC18-HPV16smE7E6 of the synthetic gene. The sequence determination showed that the synthetic sequence Consistent with design. The structure map of the cloning plasmid pUC18-HPV16smE7E6 inserted with the codon-optimized HPV16 type E7E6 fusion gene is as follows: figure 1 As shown, its sequence is shown in SQE.ID.NO.3, a total of 3475bp.

[0054] The pUC18 used in this example is a commercially available plasmid vector, which was purchased from Beijing Liuhetong Economic and Trade Co., Ltd. For its specific structure, see the literature "Guidelines for Molecular Biological Cloning", Sambrook J et al., second edition...

Embodiment 3

[0055] Example 3 : Construction of recombinant adenovirus type 5 shuttle plasmid pDC-HPV16smE7E6

[0056] This example is to construct the recombinant adenovirus type 5 shuttle plasmid pDC-HPV16smE7E6, the specific process is as follows.

[0057] First, the optimized SmE7E6 gene synthesized in Example 2 and cloned into pUC18 was digested with BglII enzyme in a 37°C water bath for 2 hours, and then the SmE7E6 fusion gene fragment was recovered with an agarose gel recovery kit.

[0058] Secondly, the SmE7E6 fusion gene fragment was inserted into the adenovirus shuttle plasmid pDC316 digested with the same endonuclease (Bgl II) and dephosphorylated with alkaline phosphatase (see figure 2 ), identified by Ava I, BglII and NsiI enzyme digestion (electrophoresis results such as Figure 4 shown) and sequenced and screened to obtain the recombinant adenovirus shuttle plasmid pDC-HPV16SmE7E6 with correct insertion, its structural map is as follows image 3 As shown, its sequence i...

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Abstract

The invention provides a gene, an expression vector, an expression method, an expression cell and an application of a human papilloma virus (HPV) 16 E7E6 fusion protein. In the invention, recombinant adenovirus is adopted to fuse E6 and E7 protein genes which have no converting activity after modification, and the mammalian cell codon optimization and transformation is performed based on the amino acid sequences of the fusion protein to lead the codon to be expressed in the mammalian cells in a high level. In the invention, an E7E6 fusion protein mammalian cell codon optimization gene is designed successfully, the recombinant adenovirus capable of expressing the HPV16 E7E6 fusion proteins in a high level is successfully constructed, has strong immunogenic performance, can induce immunoreaction of specific T cells, and completely protects immune mice from attacking of anti-tumor cells. The invention can be used for searching and developing therapeutic vaccines of cervical cancer.

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 16 E7E6 fusion protein, expression vector, method and immunotherapy application. Background technique [0002] A large number of research data prove that the high-risk human papillomavirus (humanpapillomavirus, HPV) infection of the reproductive tract is closely related to cervical cancer. HPV DNA can be found in 90% of cervical cancer patients, among which HPV16 accounts for 50%, which is the main pathogenic factor causing cervical cancer. Although the curative effect of surgery and radiotherapy is good for patients with early stage cervical cancer, radiotherapy and certain chemotherapy must be given on the basis of surgery for patients with advanced cervical cancer. Due to the huge side effects of radiotherapy and the insensitivity of cervical cancer to chemotherapy, 35% of patients still have...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N15/861C12N5/10C12P21/02A61K39/12A61K48/00A61P31/20A61P35/00C12R1/93
Inventor 田厚文任皎赵莉高见冯靖庞正吴小兵阮力
Owner 中国疾病预防控制中心病毒病预防控制所
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