摘要
目的:利用杆状病毒载体表达系统(baculovirus expression vector system,BEVS)表达重组角质细胞生长因子-1(KGF-1),并对其进行鉴定纯化及活性测定。方法:PCR扩增角质细胞生长因子-1基因(KGF-1)序列,克隆到pFastBac杆状病毒转座载体,经酶切测序鉴定出阳性重组转座载体pFastBac-kgf1后,转化含有穿梭载体Bacmid的DH10Bac感受态细胞,蓝白斑筛选出阳性质粒Bacmid-kgf1,转染Spodoptera frugiperda(sf9)昆虫细胞。对收获的蛋白进行SDS-PAGE、Westernblot鉴定。利用肝素亲和层析柱和阳离子交换柱对目的蛋白进行纯化,BaF3细胞检测细胞增殖活性,并利用C57BL/6小鼠检测促毛囊增殖活性。结果:分子量约21 kDa的KGF-1在昆虫细胞中实现了表达。病毒侵染细胞48 h时开始表达目的蛋白,到96 h时表达量达到最高;且感染复数multiplicity of infection(MOI)为4时表达量较好。经过纯化之后,蛋白纯度达到90%以上,蛋白产量达2 mg/L。纯化之后的蛋白对转染FGFR2Ⅲb的BaF3细胞在0.10 ng/ml-1.56 ng/ml之间有促增殖活性,呈剂量依赖关系。并且KGF-1处理后小鼠毛囊增殖效果有所加强。
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.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2013年第3期47-53,共7页
China Biotechnology
