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HPV16L1-h protein and coding gene and application thereof

A technology of L1 protein, pmv-05-hpv16l1-h, applied in application, gene therapy, genetic engineering, etc., can solve the problems of high production cost, inability to use, low expression level, etc., and achieve particle integrity, good safety, The effect of excellent immune characteristics and biological activity

Active Publication Date: 2014-09-10
BEIJING MINHAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Merck's preventive HPV quadrivalent vaccine (Saccharomyces cerevisiae expression system) and GSK's HPV bivalent vaccine (insect baculovirus expression system) currently on the market, due to their low expression and high production costs, the product price On the high side, it cannot be used by a wide range of people

Method used

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  • HPV16L1-h protein and coding gene and application thereof
  • HPV16L1-h protein and coding gene and application thereof
  • HPV16L1-h protein and coding gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0050] The gene sequence of embodiment 1 coding HPV16L1-h protein is optimized

[0051] According to the nucleotide sequence of the L1 protein of the highly pathogenic HPV16 strain popular in my country, the vector software was used to optimize the design of the HPV16L1 gene sequence according to the most preferred codons of Hansenula to improve its expression in Hansenula cells expression volume. The nucleotide sequence of the gene encoding HPV16L1-h protein provided by the present invention is the sequence without the yeast secretion signal peptide or the transcription termination signal recognized by the yeast. The codons of the gene encoding HPV16L1-h protein in the present invention use the most preferred codons of Hansenula. For codon usage frequencies in Pichia angusta see http: / / www.kazusa.or.jp / codon / . In order to prevent the GC content of the translated mRNA from being too high and the secondary structure of the mRNA from affecting the translation efficiency, the ...

Embodiment 2

[0052] The construction of embodiment 2 recombinant expression vector PMV-05-HPV16L1-h

[0053] 1. Construction process of expression vector PMV-05:

[0054] The expression vector PMV-05 of the present invention consists of 6 parts: promoter (MOX-P), terminator (MOX-T), replicon HARS, ura3 gene, ColE1 replicon, Amp resistance gene.

[0055] Using yeast genomic DNA as a template, primers MOXP-F (see SEQ ID NO.3 for sequence), MOXP-R (see SEQ ID NO.4); MOXT-F (see SEQ ID NO.5), MOXT-R respectively (see SEQ ID NO.6); HARS-F (see SEQ ID NO.7), HARS-R (see SEQ ID NO.8); Ura3-F (see SEQ ID NO.9), Ura3-R (see SEQ ID NO.10) was amplified by PCR to extract genes MOXP, MOXT, HARS, and Ura3. Using the PBR322 plasmid (purchased from Treasure Bioengineering Dalian Co., Ltd., item number: D3050) as a template, and using primers Amp+ColE1-F (see SEQ ID NO.11), Amp+ColE1-R (see SEQ ID NO.12) for Amplify by PCR and transfer the gene Amp+ColE1. The PCR reaction system is as follows: 10 μl o...

Embodiment 3

[0061] Example 3 Induced Expression and Detection of HPV16L1 Strain H Expressed by Hansenula

[0062] The recombinant expression vector PMV-05-HPV16L1-h was transformed into Hansenula ATCC26012 uracil-deficient host cells by electroporation (the wild-type host bacteria came from ATCC26012, and the ATCC26012 uracil-deficient host cells were obtained by gene knockout method).

[0063] ATCC26012 uracil-deficient host cells were obtained: the G418 resistance gene sequence was obtained from the Pichia pastoris expression vector pPIC9K (purchased from inritrogen), and the Hansenula Ura3 gene sequence was obtained from Gene bank. Primers P1, P2, P3, P4, P5, and P6 were designed according to the Ura3 and G418 gene sequences, and their nucleotide sequences are shown in SEQ ID NO.13-18, respectively.

[0064] Using the genomic DNA of Hansenula wild-type host strain ATCC26012 as a template, the 5' end gene fragment of Ura3 was obtained by PCR amplification with primers P1 and P2 respecti...

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Abstract

The present invention provides a human papilloma virus 16 type L1 protein HPV16L1-h and a coding gene and application thereof. The amino acid sequence of the HPV16L1-h protein is shown as SEQ ID No. 2, and the coding gene is shown as SEQ ID No. 1 in a sequence table. The present invention also provides an HPV16L1-h recombinant expression vector. The HPV16L1-h protein provided by the present invention has good immunogenicity, no potential carcinogenic risk, has good safety, immunological characteristics and biological activity, can be prepared and purified in a large scale, can be used for preparing a vaccine for preventing cervical cancer and drugs for treating cervical cancer, and has good economic values and application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to human papillomavirus type 16 L1 protein HPV16L1-h and its coding gene and application. Background technique [0002] Cervical cancer is a common invasive disease that seriously endangers women's health. Its incidence rate is second only to breast cancer, ranking second among female malignant tumors in the world, and even ranking first in some developing countries. According to the statistics of the World Health Organization (WHO), about 500,000 women worldwide suffer from cervical cancer every year, and nearly 300,000 women die from cervical cancer, of which 80% of the deaths occur in developing countries. According to statistics, there are about 130,000 cervical cancer patients in my country, and about 50,000 people die from cervical cancer every year, and the incidence rate is increasing year by year and younger. In 1995, the International Center for Cancer Research announced the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/025C12N15/37C12N15/81C12N1/19C12N7/04A61K48/00A61K39/12A61P35/00A61P31/20C12R1/78
Inventor 顾美荣宋琳琳孟凡童戚治国张凯泉魏文进刘建凯
Owner BEIJING MINHAI BIOTECH
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