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Chimeric protein vaccine containing HPV16 type L1 and E7 target antigens expressed in yeast cells and its preparation method

A yeast and vaccine technology, applied in the field of HPV vaccine, can solve the problem of inability to obtain HPV vaccine by preparation method, and achieve the effects of being suitable for large-scale production, simple operation, significant economic and social benefits

Inactive Publication Date: 2012-08-01
CHENGDU KANGHUA BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since it is currently impossible to obtain a large amount of HPV virus through tissue culture, and considering the potential carcinogenicity of HPVE6 and E7 genes, HPV vaccines cannot be obtained through traditional vaccine preparation methods such as attenuated live vaccines or inactivated vaccines

Method used

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  • Chimeric protein vaccine containing HPV16 type L1 and E7 target antigens expressed in yeast cells and its preparation method
  • Chimeric protein vaccine containing HPV16 type L1 and E7 target antigens expressed in yeast cells and its preparation method
  • Chimeric protein vaccine containing HPV16 type L1 and E7 target antigens expressed in yeast cells and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0012] (1) Treatment of clinical samples: scrape the surface cells of the cervix, centrifuge, wash and centrifuge with a suitable buffer, and precipitate for later use. If not used immediately, it must be stored in a -70°C freezer or liquid nitrogen.

[0013] (2) Refer to Genbank, isolate HPV genomic DNA from clinical samples, and determine representative HPV wild-type sequences. Consider the phenol extraction method: suspend the cells in the extract containing EDTA, SDS and RNAase, then use proteinase K and SDS to synergistically break the cell membrane, and use phenol and chloroform to denature the protein and remove impurities. Genomic DNA was finally precipitated with ethanol.

[0014] (3) Clone HPV16LI / E7 gene and construct plasmid pPIC9k-L1 / E7

[0015] a) Primer design

[0016] Primers were designed according to the full sequence of HPV16 in GenBank, 6*his tag and SnaBI restriction site were introduced upstream of L1, HindIII restriction site was introduced downstre...

Embodiment 2

[0092] Example 2 PCR identification of recombinants

[0093] Using the L1 upstream primer and the E7 downstream primer to amplify the extracted yeast genome as a template, the results were obtained, indicating that the recombinant yeast GS 115 and pPIC9k-L1 / E7 were successfully constructed.

Embodiment 3

[0094] Example 3 High copy exogenous gene integration screening and identification of recombinant yeast GS 115 positive transformants:

[0095]Select a single positive colony, extract the transformed genomic DNA of yeast GS 115 as a template according to the kit instructions, use pPIC9k-L1 / E7 to construct primers and conditions for PCR amplification screening, and analyze the PCR products by agarose gel electrophoresis with nucleic acid dyes .

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Abstract

The invention provides an L1E7 chimeric protein vaccine composed of L1 capsid protein and E7 polypeptide and a preparation method thereof. In the invention, optimized induction expression conditions are employed, and expression of a chimeric protein through large scale fermentation of yeast cells can significantly reduce the cost. Moreover, the purification process adopted in the invention is relatively simple and has low cost, thus being suitable for mass production of HPV16L1 / E7 protein. The HPV16L1 / E7 protein containing a late structural gene and an early gene can also induce the body to generate strong E7-specific C TL reaction and antitumor activity besides enabling the body to generate a neutralizing antibody. Therefore, the chimeric protein vaccine provided in the invention is considered to be the most promising vaccine for cervical cancer prevention and treatment, and can be used for HPV16 infected patients with or without clinical symptoms.

Description

Technical field: [0001] The invention belongs to the technical field of biomedicine, in particular to the technical field of biological vaccines, and relates to using chimeric proteins of HPV16L1 and HPV16E7 as target antigens and combining them in appropriate proportions to prepare HPV vaccines for preventing some diseases caused by HPV. Background technique: [0002] Cervical cancer is one of the most common malignant tumors in women, and its incidence is second only to breast cancer, ranking second among female malignant tumors. According to worldwide statistics, there are about 500,000 new cases of cervical cancer every year, and 600 women die of cervical cancer every day, 80% of which are in developing countries. There are about 131,500 new cases in my country every year, accounting for 1 / 3 of the world's new cases. A large number of research data show that high-risk human papillomavirus (HPV) infection is one of the important causes of cervical cancer. HPV is a kind ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61P31/20A61P35/00A61P15/00C12N15/81C12R1/84
Inventor 侯文礼冯晓赵志鹏刘瑾钟泽荣
Owner CHENGDU KANGHUA BIOLOGICAL PROD
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