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Method for producing recombinant hepatitis B surface antigen

A technology of hepatitis B surface antigen and production method, which is applied in the production field of recombinant hepatitis B surface antigen, can solve the problems of VLP structure change, low yield, unstable purification process, etc., and achieve stable quality, short process cycle and high recovery rate Effect

Active Publication Date: 2018-07-27
JIANGSU THERAVAC BIO PHARMA
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  • Abstract
  • Description
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Problems solved by technology

[0006] The existing recombinant HBsAg derived from various yeasts are all expressed intracellularly, and the purification processes of the manufacturers are different. The mainstream purification process includes steps such as hydrophobic chromatography. The main principle is to use the HBsAg-rich lipid , strong hydrophobic properties, but the antigen may cause a certain degree of VLP structural changes during the adsorption / desorption process with the chromatography packing material, resulting in unstable purification process and low yield

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  • Method for producing recombinant hepatitis B surface antigen
  • Method for producing recombinant hepatitis B surface antigen
  • Method for producing recombinant hepatitis B surface antigen

Examples

Experimental program
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Embodiment 1

[0044] The construction of embodiment 1 expression vector pMAUR (S.C) KARS1

[0045] The primer sequences involved in the vector construction process are as follows:

[0046]

[0047]

[0048] 1) PCR amplification to obtain the MOX promoter sequence of methanol oxidase gene

[0049] Use the Yeast Genome Extraction Kit to extract Hansenula (ATCC 14754) genomic DNA, use the genomic DNA as a template, and use primer 1 and primer 2 to perform PCR reaction. The specific reaction conditions are: 50 μl reaction system, containing 1 μl KOD-Plus polymer Enzyme, 5μl 10×Buffer, 5μl dNTP, 2μl 25mM MgSO 4 , 1.5 μl primer 1, 1.5 μl primer 2, 1 μl template, 33 μl ddH 2 O. The reaction was carried out with a Bio-Rad PCR instrument. The reaction conditions were: 94°C for 2 minutes, cycle once; 94°C for 15 seconds, 55°C for 1 minute, 68°C for 1.5 minutes, a total of 35 cycles; 68°C for 1 minute, cycle 1 Second-rate. The PCR product was recovered and purified. The target fragment wa...

Embodiment 2

[0073] Example 2 Screening of Recombinant Hepatitis B Surface Antigen Hansenula High Expression Strain

[0074] 1) Transformation and screening of recombinant expression hepatitis B surface vector

[0075] Competent cell preparation: Pick a single clone of the URA3 auxotrophic Hansenula strain and culture it overnight at 37°C in 10mL of LYPD medium. Take 2 mL of the culture, inoculate it into a shaker flask containing 100 mL of YPD medium, and culture it at 37°C until OD600=1.3-1.5. Collect the cells by centrifuging at 3000g for 10 minutes at 4°C, and suspend the cells with 0.2 times the volume of pre-cooled 50mM phosphate buffer. Incubate at 37°C for 15 minutes. Centrifuge as above, suspend and wash the cells with 1 volume of pre-cooled STM buffer. Centrifuge as above, suspend and wash the cells with 0.5 volume of pre-cooled STM buffer. Centrifuge as above, and suspend the cells with 0.005 times the volume of pre-cooled STM buffer to a final volume of about 0.4 ml.

[0...

Embodiment 3

[0079] Example 3 Hansenula expresses recombinant hepatitis B surface antigen 50L bioreactor fermentation

[0080] 1) Preparation of first-level seed liquid: Take one of the above-mentioned recombinant Hansenula glycerol bacteria liquid (1.5mL / tube) and inoculate it into 200mL YPG medium, which contains: 20.00g / L glycerol, 10.00g / L yeast extract Material, 20.00g / L soytone. Adjust the rotation speed to 220rpm and culture at 37°C for 12 hours; when the OD600 reaches 10-15 hours, use it as the primary seed solution;

[0081] 2) Secondary seed solution preparation: the above-mentioned primary seed solution was inoculated in 2.0L BSM inorganic salt medium, and the BSM inorganic salt contained: 0.90g / L CaSO 4 .2H 2 O, 11.67g / L MgSO 4 .7H 2 O, 14.67g / L K 2 SO 4 , 9.00g / L (NH 4 ) 2 SO 4 , 50g / L glycerin and 25.05g / L sodium hexametaphosphate. Add 2mL of PTM1 trace elements per liter of BSM, PTM1 formula contains: 6.00g / L CuSO 4 .5H 2 O, 3.00g / L MnSO 4 .H 2 O, 0.02g / L H ...

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Abstract

The invention relates to a method for producing a recombinant hepatitis B surface antigen. According to the invention, having undergone bidirectional pressurization screening, a bacterial strain acquired by using the method is high in copy number and genetically stable; by controlling the concentration of methanol, high-density fermentation process is stable and the level of expression is high; and the recombinant hepatitis B surface antigen with high purity and high recovery rate can be obtained by subjecting yeast cells to fragmentation, clarification, silica gel adsorption, ion exchange, ultrafiltration, concentration, density gradient centrifugation and molecular sieve chromatography after fermentation, so the method has the characteristics of short process period, high antigen purity,uniform antigen particles and the like.

Description

technical field [0001] The invention belongs to the field of protein medicine production, and in particular relates to a production method of recombinant hepatitis B surface antigen. Background technique [0002] According to the report of the World Health Organization, about 2 billion people in the world are infected with HBV, and about 650,000 people die each year from liver failure, cirrhosis and liver cancer caused by HBV infection. Of the 2 billion infected people, about 240 million are chronic HBV infections, and China accounts for about 1 / 3 (quoted from the 2015 edition of the World Health Organization Guidelines for the Prevention and Treatment of Hepatitis B). At present, there is no effective drug for the treatment of hepatitis B and HBV carriers. It is of great significance to develop and apply hepatitis B vaccine to prevent hepatitis B and treat HBV carriers and hepatitis B patients. Due to the problems of carrying HIV and other viruses and limited production, t...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12P21/02C07K14/02C07K1/36C07K1/34C07K1/18C07K1/16C12R1/78
CPCC07K14/005C12N15/815C12N2730/10122C12P21/02
Inventor 李建强任苏林孙洪林周童顾月葛君戚凤春鲍梦汝韩杰陈晓晓
Owner JIANGSU THERAVAC BIO PHARMA
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