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Method for constructing eukaryon Hansenula polymorpha engineering bacteria with recombinant hepatitis B virus genes and method for producing hepatitis B surface antigens

A technology of surface antigen and Hansenula, applied in genetic engineering, virus/bacteriophage, biochemical equipment and methods, etc.

Active Publication Date: 2016-07-06
ANHUI ZHIFEI LONGCOM BIOPHARM CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no specific drug for the treatment of hepatitis B, the most effective means is vaccination against hepatitis B

Method used

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  • Method for constructing eukaryon Hansenula polymorpha engineering bacteria with recombinant hepatitis B virus genes and method for producing hepatitis B surface antigens
  • Method for constructing eukaryon Hansenula polymorpha engineering bacteria with recombinant hepatitis B virus genes and method for producing hepatitis B surface antigens
  • Method for constructing eukaryon Hansenula polymorpha engineering bacteria with recombinant hepatitis B virus genes and method for producing hepatitis B surface antigens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Cloning of Hansenula methanol oxidase promoter and terminator

[0045] According to the known sequences (GenBank: A11156, AR363832 and E00783), primers MOX_P-F and MOX_P-R located at both ends of the MOX gene promoter were synthesized, respectively,

[0046] Wherein MOX_P-F is 5'-CCAATAGATCTTCGACGCGGAGAACGATCTCCTC-3',

[0047] MOX_P-R1 is 5'-GGAACCTCCACCAACAACAATGATATCGAAT-3', and the primers MOX_TT-F1 and MOX_TT-R1 located at both ends of the MOX gene terminator are synthesized,

[0048] Where MOX_TT-F1 is 5'-TCGGAACTTACGAGGAGACCGGACTTGCCAG-3',

[0049] MOX_TT-R1 is 5′-CTTGTGTCTCACACCCATAATGATCCCGTT-3′, using Hansenula genomic DNA as a template (for the genome extraction method, see page 485 of "Molecular Cloning Experiment Guide, Third Edition"), the MOX promoter and terminator were amplified by PCR , the amplified fragment was directly inserted into the pEASY-Blunt-Zero plasmid, and according to the method provided by the company, the bacterial clone con...

Embodiment 2

[0050] Embodiment 2: Construction of expression vector pHPZF1.0

[0051] The MOX-P promoter was amplified by PCR from the pMOX-P plasmid, and cloned into the vector pPICZC using the BglII-EcoRI site to obtain the plasmid pPICZC-MOXP; according to the sequence of the MOX promoter obtained by sequencing verification, the primer MOX_P-R2 was redesigned. With MOX_P-F and MOX_P-R2 as primers,

[0052] Where MOX_P-F is 5'-CCAATAGATCTTCGACGCGGAGAACGATCTCCTC-3' (the underlined part is the BglII site), MOX_P-R2 is 5'-CACGTGAATTCCTCGTTTCGAAGCTTTGTTTTTGTACTTTAGATT-3' (the underlined part is the EcoRI site), and the intermediate plasmid vector pMOX-PDNA As a template (for the plasmid extraction method, refer to page 485 of "Molecular Cloning Experiment Guide, Third Edition"), the MOX promoter was amplified by PCR, and BglII and EcoRI sites were introduced at both ends of the PCR product. Double digestion was performed with restriction endonuclease BglII-EcoRI, and the DNA fragment was se...

Embodiment 3

[0056] Example 3: Analysis of the consensus amino acid sequence of adr subtype HBsAg

[0057] Since genotype C includes all adr serotypes, we used the standard genome sequence AY123041 of hepatitis B virus type C reported in Japan as the search sequence, and the BlastNCBI nucleic acid database (similarity parameters were set to 93-100%), and a total of nearly 2000 pieces. After investigation, 617 HBV genome sequences of genotype C reported in China were sequenced. Excluding sequencing errors and HBsAg nonsense mutation sequences, a total of 479 HBsAg protein sequences of adr serotype reported in China were obtained (25 of which were from Hong Kong sequences and 4 of them were from Hong Kong). from Taiwan). Use the function of BioEdit software for amino acid sequence comparison analysis to obtain the most representative HBsAg consensus amino acid sequence (consensusaminoacidsequence, that is, the sequence of amino acid residues with the highest probability of occurrence at eac...

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Abstract

The invention discloses a method for constructing eukaryon Hansenula polymorpha with recombinant hepatitis B virus genes and a method for producing hepatitis B surface antigens, and belongs to the technical field of bioengineering. The method for constructing the eukaryon Hansenula polymorpha includes steps of 1, constructing plasmids pHPZF1.0-ZS with the recombinant hepatitis B virus HBsAg genes; 2, screening the plasmids to obtain the eukaryon Hansenula polymorpha engineering bacteria with the recombinant hepatitis virus HBsAg genes and the highest expression level. The methods have the advantages that HBsAg recombinant proteins can be stably and efficiently expressed in a methanol induction mode by Hansenula polymorpha engineering bacterial strains of the hepatitis B surface antigens which can be obtained by the aid of the methods, and the methods are suitable for producing the HBsAg recombinant proteins on a large scale.

Description

technical field [0001] The invention relates to the construction of eukaryotic Hansenula engineering bacteria containing recombinant hepatitis B virus gene and the production method of hepatitis B surface antigen, belonging to the technical field of bioengineering. Background technique [0002] Hepatitis B (referred to as hepatitis B) vaccine has been widely used for more than 20 years, but hepatitis B virus (HepatitisBvirus, HBV) infection is still one of the most serious global health problems. According to the report of the World Health Organization, about 2 billion people in the world have current or past signs of hepatitis B virus infection, and about 600,000 people die every year from liver failure, cirrhosis and hepatocellular carcinoma caused by HBV infection. There are more than 100 million hepatitis B virus carriers in my country, and more than 20 million existing patients. The epidemic in the south is heavier than that in the north, and the rural areas are heavier...

Claims

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Application Information

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IPC IPC(8): C12N15/51C12N15/81C12N1/19C07K14/02
CPCA61K39/00C07K14/005C12N15/815C12N2730/10122C12N2730/10151C12N2800/102
Inventor 黄恩启吴常伟李超王力卫刘术敏程英杰陶立峰杨世龙
Owner ANHUI ZHIFEI LONGCOM BIOPHARM CO LTD
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