Recombinant DNA sequence, hansenula polymorpha, preparation method for hepatitis B surface antigen, and hepatitis B vaccine

A technology of DNA sequence and surface antigen, which is applied in the field of genetic engineering to achieve high yield, high genetic stability and high expression level

Inactive Publication Date: 2014-12-24
北京天坛生物制品股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the recombinant hepatitis B vaccines produced in Europe, America and India are of the adw subtype, and there are no reports at home and abroad of using Hansenula to express adr to produce hepatitis B vaccines

Method used

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  • Recombinant DNA sequence, hansenula polymorpha, preparation method for hepatitis B surface antigen, and hepatitis B vaccine
  • Recombinant DNA sequence, hansenula polymorpha, preparation method for hepatitis B surface antigen, and hepatitis B vaccine
  • Recombinant DNA sequence, hansenula polymorpha, preparation method for hepatitis B surface antigen, and hepatitis B vaccine

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preparation example Construction

[0040] The present invention also provides a method for preparing the surface antigen of the adr subtype hepatitis B virus, which comprises: fermenting and culturing the recombinant yeast as described in any one of the above items, so as to obtain the surface antigen of the adr subtype hepatitis B virus.

[0041] Preferably, the method further includes: after fermenting and culturing the recombinant yeast, performing cell disruption, filtration, adsorption, and hydrophobic chromatography, so as to prepare further purified adr subtype hepatitis B virus surface antigen.

[0042] Preferably, the filtration is ultrafiltration; the adsorption is silica gel adsorption; and the hydrophobic chromatography is butyl agarose hydrophobic chromatography.

[0043] The present invention also provides a hepatitis B vaccine, characterized in that the vaccine comprises the surface antigen of the adr subtype hepatitis B virus obtained by any one of the above-mentioned preparation methods.

[004...

Embodiment 1

[0046] Obtaining of embodiment 1 recombinant DNA sequence

[0047] According to the scientific literature "A.K.Raney and A.McLachlan, The Biology of Hepatitis B Virus, In: Editor A.Mclachlan ed. Molecular Biology of the Hepatitis B Virus, 2000, CRC Press." Obtain the adr subtype hepatitis B virus surface antigen amino acid sequence. The above nucleic acid sequence encoding HBsAg was redesigned with reference to the codon frequency of the Hansenula gene (see US Patent No. 5,712,114).

[0048] During the design, according to the amino acid sequence, the Hansenula optimal coding for the adr subtype hepatitis B virus surface antigen was artificially obtained from the codon frequency of the Hansenula gene (see US patent document US5712114), and the restriction endonuclease site was searched with DNAMAN software point, excluding BamHI and EcoRI restriction sites.

[0049] According to the above design, a sequence composed of 678 bases was obtained, as shown in SEQ ID NO: 1 in the ...

Embodiment 2

[0050] Example 2 Construction of recombinant Hansenula polymorpha

[0051] The pMPT-02 plasmid (obtained from Tianjin Bohui Biotechnology Co., Ltd.) was digested with EcoRI and BamHI (the total volume of the reaction system: 50 μl, including 5 μl of the B buffer system of Roche Reagent Company, EcoRI 1.5 μl; BamHI 1.5 μl, pMPT- 02 plasmid 10 μl, temperature is 37°C, reaction time is 1 hour), electrophoresis to recover the digested vector fragment, use the DNA recovery kit of Treasure Bio (Dalian) Co., Ltd. and recover the digested vector fragment according to its instructions) According to the steps described in the appendix of TaKaRa PCR Ligation Kit (Code No.D6022, obtained from Treasure Bioengineering (Dalian) Co., Ltd.), the vector fragment was connected to the insert fragment shown in SEQ ID NO: 3 described in Example 1 Afterwards, thermal transformation was carried out, and the thermal transformation conditions were as follows: 5 μl of the ligated recombinant plasmid was...

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Abstract

The invention provides a recombinant DNA sequence, hansenula polymorpha, a preparation method for hepatitis B surface antigen, and a hepatitis B vaccine. The recombinant DNA sequence is obtained by codon optimization of coding genes of the hepatitis B virus surface antigen according to codon usage frequency of the hansenula polymorpha. The invention also provides the hansenula polymorpha comprising the recombinant DNA sequence, a method for preparing adr sub-type hepatitis B surface antigen by using the recombinant DNA sequence, and the hepatitis B vaccine. The adr sub-type hepatitis B surface antigen has high expression level of the recombinant DNA sequence. The recombinant hansenula polymorpha is fast in growth speed, has high HBsAg yield, can be fermented in high cell density by using a cheap chemically synthetic medium, has low fermentation contamination rate and is beneficial to large-scale production; and HBsAg adr vaccine provided by the invention has high trend of Th1 and Th2 type cellullar immunologic response.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular, the invention relates to a recombinant DNA sequence of adr subtype hepatitis B surface antigen (HBsAg) and its application. Background technique [0002] China is a high-incidence area of ​​hepatitis B, and the carrier rate of hepatitis B virus surface antigen (HBsAg) reaches about 10%. The current method of controlling hepatitis B mainly relies on prevention, and hepatitis B vaccine is the most effective weapon to prevent hepatitis B. By separating and purifying HBsAg subviral particles from the blood of hepatitis B patients, the first generation of blood-derived polypeptide hepatitis B vaccine was successfully developed. Later, using the simplest eukaryotic cell - Saccharomyces cerevisiae (Saccharomyces cerevisiae) as a recipient cell, people successfully constructed a DNA recombinant HBsAg engineering strain. It has become the raw material for the development of the second-g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/51C12N1/19C12P21/02A61K39/29A61P31/20C12R1/93C12R1/78
Inventor 李津许毅张德有杨云凯王曦马锐
Owner 北京天坛生物制品股份有限公司
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