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Hansenula polymorpha specific expression vector construction method and method for enhancing expression quantity of hepatitis B surface antigen on hansenula polymorpha

An expression vector, the technology of Hansenula yeast, applied in the field of bioengineering

Active Publication Date: 2016-07-13
ANHUI ZHIFEI LONGCOM BIOPHARM CO LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Hansenula polymorpha specific expression vector construction method and method for enhancing expression quantity of hepatitis B surface antigen on hansenula polymorpha
  • Hansenula polymorpha specific expression vector construction method and method for enhancing expression quantity of hepatitis B surface antigen on hansenula polymorpha
  • Hansenula polymorpha specific expression vector construction method and method for enhancing expression quantity of hepatitis B surface antigen on hansenula polymorpha

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Cloning of Hansenula methanol oxidase promoter and terminator

[0035] 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,

[0036] Wherein MOX_P-F is 5'-CCAATAGATCTTCGACGCGGAGAACGATCTCCTC-3',

[0037] 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,

[0038] Where MOX_TT-F1 is 5'-TCGGAACTTACGAGGAGACCGGACTTGCCAG-3',

[0039] 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

[0040] Embodiment 2: Construction of expression vector pHPZF1.0

[0041] 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,

[0042] 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

[0046] Example 3: Analysis of the consensus amino acid sequence of adr subtype HBsAg

[0047] 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 hansenula polymorpha specific expression vector construction method and a method for enhancing expression quantity of the hepatitis B surface antigen on hansenula polymorpha and belongs to the technical field of bioengineering. The hansenula polymorpha specific expression vector construction method includes the steps of 1, designing specific primers from a eukaryotic hansenula polymorpha strain ATCC34438 or derivative strain genome DNA thereof prior to PCR (polymerase chain reaction) amplification, and retrieving a promoter MOXp and a terminator MOX-TT of an MOX gene; 2, substituting the promoter MOXp and the terminator MOX-TT of the MOX gene for a promoter AOX1 and a terminator AOX-TT on a pichia pastoris expression vector pPICZC respectively so as to obtain a hansenula polymorph expression vector pHPZF1.0.By the methods, the hepatitis B surface antigen hansenula polymorpha engineering strain is capable of expressing HBsAg (hepatitis B surface antigen) recombinant proteins stably and efficiently by means of methanol induction and is applicable to large-scale production of the HBsAg recombinant proteins.

Description

technical field [0001] The invention relates to the construction of a Hansenula specific expression vector and a method for increasing the expression level of hepatitis B virus surface antigen in Hansenula, 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 than t...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/51C12R1/78
CPCC07K14/005C12N15/815C12N2730/10122C12N2800/102C12N2800/22C12N2830/702
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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