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Method for producing recombinant human serum albumin-interferon alpha 2b

A technology of human serum albumin and human serum albumin, which is applied in the field of recombinant DNA technology to produce genetically engineered protein drugs, can solve the problems of virus suppression, restriction, and short half-life

Active Publication Date: 2013-08-07
山东泉港药业有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Ordinary interferon (IFN) has a short half-life in the body, and interferon is quickly decomposed. During the treatment period, it cannot always maintain an effective blood drug concentration and permanently inhibit the virus, so that IFN is effective in treating hepatitis C virus (HCV). ) infection potential is limited

Method used

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  • Method for producing recombinant human serum albumin-interferon alpha 2b
  • Method for producing recombinant human serum albumin-interferon alpha 2b
  • Method for producing recombinant human serum albumin-interferon alpha 2b

Examples

Experimental program
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Effect test

Embodiment Construction

[0025] 1. Construction of recombinant human serum albumin-interferon α2b Pichia pastoris engineering strain

[0026] 1. Acquisition of the target gene

[0027] According to the sequence information of the HSA gene published in GeneBank, design PCR primers

[0028] XhoI-HSA-P5: 5'-cat ctc gag (endonuclease XhoI site) aaa aga (Kex2 protease cleavage site) gat gca cac aag agt gag gtt-3'

[0029] BamHI-HSA-P3: 5'-cat gga tcc (BamHI endonuclease site) taa gcc taa ggc agc ttgact tgc agc-3'

[0030] According to the sequence information of plasmid pHC16IFN-α2b from the existing interferon α2b engineering bacteria, design PCR primer BamHI-2b-P5: 5'-cat gga tcc (endonuclease BamHI site) tgt gat ctg cct caa accca-3'

[0031] NotI-2b-P3: 5'-cat gcggccgc (NotI site of endonuclease) tca ttc ctt act tct taaact ttc-3'

[0032] Use primers XhoI-HSA-P5 and BamHI-HSA-P3 to amplify the human HSA gene from the cDNA first-strand library, and XhoI-HSA-P5 primers add restriction endonuclease XhoI...

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Abstract

The present invention relates to a method for producing recombinant human serum albumin-interferon alpha 2b of secretory expression in Pichia pastoris system, particularly to efficient expression of recombinant human serum albumin-interferon alpha 2b in Pichia pastoris and expression product purification. In the present invention, Pichia pastoris engineering bacteria containing recombinant human serum albumin-interferon alpha 2b are fermented in high density, fermented liquid is subsequently centrifuged by a high-speed centrifugal machine, and fermented supernatant liquid is collected and purified by cation exchange chromatography, hydrophobic layer chromatography and molecular sieve chromatography in a combined mode. Recombinant human serum albumin-interferon alpha 2b fusion protein produced meets the regulations of Chinese Pharmacopoeia. The present invention has the advantages of high fermentation expression rate, reasonable purification technical combination, simplified operation procedure, high yield and low production cost. The present invention meets industrial requirements.

Description

technical field [0001] The invention relates to a method for producing genetically engineered protein medicine by recombinant DNA technology, in particular to the high-efficiency expression of recombinant human serum albumin-interferon alpha 2b in Pichia pastoris and the purification of the expressed product. Background technique [0002] Ordinary interferon (IFN) has a short half-life in the body, and interferon is quickly decomposed. During the treatment period, it cannot always maintain an effective blood drug concentration and permanently inhibit the virus, so that IFN is effective in treating hepatitis C virus (HCV). ) infection potential is limited. To overcome the above shortcomings, various types of long-acting IFN have been developed, such as polyethylene glycol IFN, albumin IFN and IFN liposomes. Among them, human albumin-interferon is one of the hotspots of new research, which has greater advantages in prolonging the half-life and being used for glycosylation mod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12R1/84
Inventor 刘宁刘红军宋磊
Owner 山东泉港药业有限公司
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