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中国生物工程杂志

China Biotechnology
China Biotechnology  2013, Vol. 33 Issue (3): 47-53    DOI:
    
Expression and Purification of Biological-active Recombinant Human Keratinocyte Growth Factor-1 Base on Baculovirus Expression Vector System
ZHU Xiao-jing, JIANG Chao, XUE Ping, WANG Xiao-yan, XU Dan, NAN Jia, AI Jun, LI Xiao-kun
Wenzhou Medical College, Key Laboratory of Biotechnology and Pharmaceutical Engineering of Zhejiang Province, Wenzhou 325035,China
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Abstract  Objective: the keratinocyte growth factor-1 (KGF-1) was expressed base on baculovirus expression vector system (BEVS). Methods: KGF-1 gene was amplified by PCR and then the product was cloned into the pFastBac vector at the NotⅠ and PstⅠ sites. The recombinant plasmid pFastBac-kgf1 was identified by enzyme digestion and sequencing. The recombinant pFastBac-kgf1 was transformed into E coli DH10Bac cells which contains a baculovirus shuttle vector (bacmid) and a helper plasmid. The recombinant Bacmid-kgf1 was identified by Blue-white selection and then transfected sf9 insect cells. P3 baculoviral stock was used to product KGF-1 protein at varying MOIs (1,2,4,8,16) and different times after infection (24,36,48,60,72,84,96,120 hours after infection). The protein KGF-1 was identified by SDS-PAGE and Western Blot and purified with heparin affinity column and cation-exchange column. And its activity was analyzed by BaF3 cells. Conclusion: After infection happened 48 h, KGF-1 was expressed by sf9 cells and at 96 h the expression yield reached the highest level. And the optimum multiplicity of infection (MOI) was 4 pfu/cell. The purity of KGF-1 was more than 90% and the protein production was about 2 mg/L after purification. Mitogenic activity assay demonstrated that KGF-1 could significantly stimulate the proliferation of mouse BaF3 cells in a dose-dependent manner, ranging from 0.1 to 1.56 ng/ml. Besides, subcutaneously injection of KGF-1 could significantly stimulated the growth of hair follicles.

Key wordsKeratinocyte growth factor-1      sf9 insect cells      Baculovirus expression vector system     
Received: 20 November 2012      Published: 25 March 2013
ZTFLH:  Q78  
Cite this article:

ZHU Xiao-jing, JIANG Chao, XUE Ping, WANG Xiao-yan, XU Dan, NAN Jia, AI Jun, LI Xiao-kun. Expression and Purification of Biological-active Recombinant Human Keratinocyte Growth Factor-1 Base on Baculovirus Expression Vector System. China Biotechnology, 2013, 33(3): 47-53.

URL:

https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2013/V33/I3/47

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