重组类人Ⅰ型胶原蛋白肽在大肠杆菌中的表达纯化及功能鉴定  

Expression, Purification, and Functional Characterization of Recombinant Human-like Type Ⅰ Collagen Peptide in E.coli

作　　者：杨晶[1] 余洁莹 王蒙[1] 吴少敏[1] 马毅[1] 王菊芳[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《现代食品科技》2016年第2期60-65,共6页Modern Food Science and Technology

基　　金：国家自然科学基金资助项目(21306055);高等学校博士学科点专项科研基金(20130172120041);中央高校基本科研业务费(2013M0059)

摘　　要：本研究构建了编码重组类人Ⅰ型胶原蛋白肽基因的原核表达载体pET28a-rhCⅠ,并将其转化到大肠杆菌Rosetta(DE3)中进行诱导表达。采用PCR的方法扩增重组类人Ⅰ型胶原蛋白肽的cDNA序列,并将其克隆到原核表达载体pET28a上;重组质粒转化到大肠杆菌Rosetta(DE3)中进行IPTG诱导表达,并优化表达条件。利用SDS-PAGE和WesternBlot检测表达产物。大量表达重组蛋白,采用镍亲和层析进行纯化,并对纯化后的蛋白进行体外抗氧化研究以及促细胞增殖分析。结果表明,通过大肠杆菌Rosetta(DE3)诱导表达获得了分子量约为40ku的重组蛋白,与预期相符。经镍亲和层析获得纯度较高的蛋白,通过DPPH实验证明其有一定的抗氧化活性;而且MTT实验证实该蛋白可以促进小鼠成纤维细胞3T3细胞的增殖,为其在食品、化妆品和医疗行业的应用提供一定的理论依据。A prokaryotic expression vector, pET28a-rhC I was constructed, with recombinant, human-like type I collagen peptide gene and transformed into Escherichia coli Rosetta（DE3） for inducible expression. The c DNA sequence of recombinant human-like collagen type I peptide was amplified by polymerase chain reaction（PCR） and cloned into the prokaryotic expression vector, p ET28 a. The recombinant plasmid was transformed into E. coli Rosetta（DE3） for IPTG-induced expression and gene expression conditions were optimized. The expressed products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） and western blot. The expressed recombinant proteins were purified by nickel affinity chromatography. Additionally, in vitro antioxidant activity and cell proliferation assay were conducted on the purified proteins. The results showed that the molecular weight of the expressed recombinant protein induced by of E. coli Rosetta（DE3） was approximately 40 ku, consistent with the expected value. Nickel affinity chromatography produced a collagen peptide with relatively high purity, and the 2,2-diphenyl-1-picrylhydrazyl（DPPH） free-radical test revealed that the recombinant protein showed antioxidant activity. In addition, the methylthiazolydiphenyl-tetrazolium bromide（MTT） assay showed that the recombinant protein promoted the proliferation of mouse embryonic fibroblasts cells（3T3）. These results indicate potential applications of this protein in industries such as food, cosmetics, and medicine.

关 键 词：胶原蛋白 重组表达 抗氧化 细胞增殖 

分 类 号：TS201.3[轻工技术与工程—食品科学]