HPV16L1 polynucleotide sequence and expression vector, host cell and application thereof

A HPV16L1, polynucleotide technology, applied in the direction of application, introduction of foreign genetic material using vectors, chemical instruments and methods, etc., can solve problems such as acceptance by the general public

Inactive Publication Date: 2012-07-18
王昌华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The human papillomavirus vaccines developed by Merck using brewer's yeast and GlaxoSmithKline using insect cell-b

Method used

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  • HPV16L1 polynucleotide sequence and expression vector, host cell and application thereof
  • HPV16L1 polynucleotide sequence and expression vector, host cell and application thereof
  • HPV16L1 polynucleotide sequence and expression vector, host cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: L1 gene sequence optimization

[0075] Convert the full-length 506 amino acids of the HPV16L1 amino acid sequence into the nucleotide sequence of Hansenula's preferred codon pattern, and the codon with Hansenula's preference for the first codon in the preferred demerging codons is mainly used in the Hansenula codon. Yeast prefers the first and second codons, and in a few cases, the third codon is used to complete local adjustments. DNAMAN software is used to avoid excessively long complementary sequences and repetitive sequences, and RNA structure prediction is used to avoid excessively long and complex hairpin structures. , to exclude strong secondary structures and increase the internal stability of specific molecules. The nucleotide sequence of the gene also avoids sequences that may adversely affect gene transcription and translation efficiency, such as intron splicing sequences, transcription termination sequences, internal promoter sequences (TATA) and ...

Embodiment 2

[0077] Embodiment 2: HPV16L1 gene and HPV16L1 mutant gene expression vector construction

[0078] The DNA sequence shown in Sequence 2 in the Sequence Listing was synthesized by a fully artificial method, and recognition sites for restriction endonucleases EcoR I and BamH I were added to both ends of the sequence. The synthesized DNA sequence was digested with EcoR I and BamH I, and the digested fragment was recovered by electrophoresis, and stored at -20°C for future use.

[0079] Using the genomic DNA of Hansenula polymorpha CGMCC 2.2497 as a template, clone the 1.5kb methanol oxidase gene (MOX) promoter, the 350bp methanol oxidase gene (MOX) terminator, and the 1.0kb self-replicating sequence HARS; and from YIp5 (GeneBank Accession NO.L09157) plasmid cloned the 1.1kb Saccharomyces cerevisiae uracil gene ScURA3; after connecting the above four parts, it was inserted into the multiple cloning site of the pBluescrip II plasmid to construct the shuttle vector pMPT-02.

[0080]...

Embodiment 3

[0085] Example 3: Electrotransformation of Hansenula Competent Host Cells with Expression Vectors

[0086] The vector pMPT-HPV16L1 was digested with Sac I to linearize the vector. The linearized vector was transformed into Hansenula polymorpha (Hansenula polymorpha) CGMCC NO.1218 by electroporation method (LN-101 gene pulse introduction instrument produced by Tianjin University of Technology, 1.5kV, 50μF, 200Ω, 3-5mSec). Transformants were selected on a medium plate containing 1.34g / 100ml YNB (yeast nitrogen base medium), 2g / 100ml glucose.

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Abstract

The invention relates to an HPV16L1 polynucleotide sequence, and an expression vector, a host cell and application of the HPV16L1 polynucleotide sequence. The HPV16L1 polynucleotide sequence comprises an amino acid sequence of recombinant human papillomavirus (HPV) L1 capsid protein, a synthetic nucleotide sequence coding the amino acid sequence, and a recombinant expression vector and a hansenula polymorpha expression host strain comprising the nucleotide sequence. The invention also relates to the application of HPV16L1 protein consisting of the amino acid sequence and derivatives of the HPV16L1 polynucleotide sequence in preparing vaccine. According to the invention, by transforming the nucleotide sequence of HPV16L1 wild type virogene, the recombinant HPV16L1 capsid protein in a hansenula polymorpha system is efficiently expressed, and the HPV16L1 capsid protein can be industrially produced by using the hansenula polymorpha expression system; and compared with the conventional other eukaryotic expression systems, the hansenula polymorpha expression system has the advantages of high yield, low cost and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular relates to an HPV16L1 polynucleotide sequence, and also includes an expression vector, a host cell and its polynucleotide sequence, and the application of the expression vector and the host cell in pharmaceuticals and preparations. Background technique [0002] Infection with papillomaviruses has been found in a variety of species including sheep, dogs, rabbits, monkeys, cattle and humans. Human papillomavirus (HPV for short) has been separated into hundreds of types. If the identity of the L1 gene and the L1 sequence of the previously identified type is less than 90%, then this type can be determined as a new type. A subtype is defined if the L1 amino acid sequence identity is between 90 and 98%, and a variant [compared to the prototype (parental type)] if the identity is above 98%. [0003] Human papillomavirus is a kind of DNA virus with strict host range and tissue specificit...

Claims

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Application Information

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IPC IPC(8): C12N15/37C07K14/025C12N15/81C12N1/19A61K39/12A61P31/20C12R1/78
Inventor 王昌华杨珺齐怀丰李建王艳侠汪志良张涛宋立娜王玉香孙长海汪和睦
Owner 王昌华
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