摘要
为研究重组人碱性成纤维细胞生长因子(human basic fibroblast growth factor, bFGF)的原核表达及纯化条件,并分析其功能,该研究根据bFGF基因序列,优化密码子,构建pE T-28a原核表达载体,经IPTG诱导, SDS-PAGE电泳分析验证,采用Ni柱进行分离纯化目的蛋白bFGF。培养NIH3T3细胞、HEK293细胞及CHO细胞,并进行CCK-8实验检测蛋白活性。结果显示,成功构建原核表达载体。经电泳分析,在1 mmol/L IPTG诱导条件下,成功表达目的蛋白bFGF,表达量约占菌体蛋白量32%,蛋白纯度约为96%。活性检测结果显示, ED50分别为5.97 ng/mL、4.21 ng/mL、6.71 ng/mL,可以有效促进NIH3T3细胞、HEK293细胞及CHO细胞增殖。该研究得出,经原核表达系统成功表达人bFGF蛋白且其活性较高。
In the present study,we studied the prokaryotic expression and purification of recombinant human basic fibroblast growth factor(bFGF),and analyzed its functions.Firstly,according to the bFGF gene sequence,the codon was optimized and the prokaryotic expression vector of pET-28 a was constructed.And protein expression was induced by IPTG.Secondly,The expression of bFGF was determined by SDS-PAGE electrophoresis and confirmed by SDS-PAGE.Finally,NIH3T3 cells,HEK293 cells and CHO cells were cultured and CCK-8 assay was performed to detect protein activity.The results showed that the prokaryotic expression vector was successfully constructed.The target protein bFGF was successfully expressed under the induction of 1 mmol/L IPTG.The expression amount was about 32%of the bacterial protein and the protein purity was about 96%.The results of the activity test showed that the ED50 was 5.97 ng/mL,4.21 ng/mL,and 6.71 ng/mL,which could effectively promote the proliferation of NIH3T3 cells,HEK293 cells,and CHO cells.This study demonstrates that the human bFGF protein was successfully expressed by prokaryotic expression system and its activity was high.
作者
窦媛媛
林艳
高向征
王燕芳
王小引
王天云
Dou Yuanyuan;Lin Yan;Gao Xiangzheng;Wang Yanfang;Wang Xiaoyin;Wang Tianyun(Department of Biochemistry and Molecular Biology,Xinxiang Medical University,Xinxiang453000,China;International JointResearch Laboratory for Recombinant Pharmaceutical Protein Expression System of Henan,Xinxiang453000,China;Department ofcommunity nursing,School of Nursing,Xinxiang Medical University,Xinxiang453000,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2019年第7期1365-1370,共6页
Chinese Journal of Cell Biology
基金
河南省高校科技创新团队项目(批准号:18IRTSTHN027)资助的课题~~