重组Ⅰ型人源化胶原蛋白“福活因子”的生物合成及其结构表征研究  

Biosynthesis and structural characterization of recombinant type I humanized collagen"Fuhuo Factor"

作　　者：全冰华 王勇 邓宏鸿 庞晓明 滕永慧 李璐 方伟 QUAN Binghua;WANG Yong;DENG Honghong;PANG Xiaoming;TENG Yonghui;LI Lu;FANG Wei(Guangxi Fulaiming Biopharmaceutical Co.,Ltd.,Nanning,Guangxi 530031,China;Chinese Academy of Biomedicines,Shenzhen,Guangdong 518001,China;School of Pharmacy,Zhejiang University,Hangzhou,Zhejiang 310058,China;Department of Stomatology,Zhongnan Hospital,Wuhan University,Wuhan,Hubei 430062,China)

机构地区：[1]广西福莱明生物制药有限公司,广西南宁530031 [2]中华生物医药研究院,广东深圳518001 [3]浙江大学药学院,浙江杭州310058 [4]武汉大学中南医院口腔科,湖北武汉430062

出　　处：《医药前沿》2024年第1期1-3,7,共4页Journal of Frontiers of Medicine

基　　金：中华生物医药创新基金资助项目(No.IFCABM20210606FLM03)。

摘　　要：目的:筛选获得含胶原蛋白保守序列hCol.1基因的重组人源化Ⅰ型胶原蛋白高效表达基因工程菌和高产创新融合蛋白,并通过检测其理化特征验证表达产物。方法:将His6-SUMO-hCol.1重组基因大肠杆菌高密度发酵后经镍亲和层析和离子交换层析两步纯化浓缩获得重组蛋白,检测理化测试特征。结果:测序证明克隆基因与hCol.1序列高度一致,Blast比对蛋白序列与人Ⅰ型胶原蛋白高度同源,折叠结构与人Ⅲ型胶原蛋白高度类似。表观分子量55~60kD,产量36.4mg/L,纯度>95%。等电点检测、氨基酸组成和圆二色谱等实验结合高级结构预测分析,证明其理化性状与蛋白结构符合胶原蛋白特征。结论:成功将人Ⅰ型胶原蛋白保守功能结构域hCol.1与SUMO等肽段重组融合表达并命名为福活因子(FHTFF),筛选出基因工程菌pET21a(+)-FHTFF/BL21,纯化产物“福活因子”的理化特性、序列和结构与人Ⅰ、Ⅲ型胶原蛋白肽段高度相似,属于重组人源化胶原蛋白新生物材料。Objective To screen and obtain recombinant humanized type I collagen containing collagen conserved sequence hCol.1 gene with high efficiency expression of genetically engineered bacteria and high yield of innovative fusion proteins,and to validate the expression products by detecting their physicochemical characteristics.Methods The recombinant protein was obtained by two-step purification and concentratded by nickel affinity chromatography and ion-exchange chromatography after high-density fermentation of Escherichia coli with the recombinant gene of His6-SUMO-hCol.1,and the physicochemical characteristics were detected.Results Sequencing proved that the cloned gene was highly consistent with the sequence of hCol.1,and the Blast of protein sequence was highly homologous to human collagen type I,and the folding structure was highly similar to that of human collagen type III.The apparent molecular weight was 55-60 kD,yield 36.4 mg/L,purity>95%.The experiments of isoelectric point detection,amino acid composition and circular dichroism combined with advanced structure prediction analysis proved that the physicochemical properties and protein structure were consistent with collagen characteristics.Conclusions The conserved functional structural domain hCol.1 of human type I collagen was successfully expressed in recombinant fusion with peptides such as SUMO,and named as Fuhuo Factor(factor of human thriving and flourishing,FHTFF),the genetically engineered bacterium pET21a(+)-FHTFF/BL21 was screened,and the physicochemical properties,sequences,and structures of the purified product"Fuhuo Factor"are highly similar to those of human type I and III collagen peptides,and it belongs to the new biomaterials of recombinant humanized collagen.

关 键 词：重组Ⅰ型人源化胶原蛋白 福活因子 基因工程菌 

分 类 号：R94[医药卫生—药剂学]