重组胶原蛋白多肽在大肠杆菌中的表达优化

Optimizing Expression of Recombinant Collagen Peptide in Escherichia coli

以Ⅵ型人胶原蛋白A2链基因为模板,PCR扩增得到目的基因CP6,构建了重组胶原蛋白表达载体,然后筛选高效表达的宿主菌,检测重组质粒的不稳定性以及对重组菌培养条件进行优化。结果表明,重组胶原蛋白在Rosetta(DE3)中表达量相对最高,重组质粒稳定性也较高。较佳的表达条件为接种量4%或5%,37℃、200 r/min培养至OD600 nm为0.7时,加入0.5 mol/L IPTG,培养5 h,并且纯化的重组蛋白与小鼠抗人COL6A2的单克隆抗体有特异性反应。

Designed primers to obtain the target gene （CP6） fragment through PCR,the template contained the protein polypeptide gene of Ⅵ type human collagen,and constructed the recombinant plasmid,and then screened the host bacteria of high efficient expression,detected the instability of recombinant plasmid,as well as optimized the culture conditions.The results showed that the best host bacterium was Rosetta （DE3） and the recombinant plasmid has higher stability. The optimal expression conditions were inoculum of 4% and 5%,37℃,200 r/min,cultured to OD600 nm 0.7 and added 0.5 mol/L IPTG to the culture medium,and then cultured 5 h. Western blotting showed that the expressed protein could specifically bind with human monoclonal antibody COL6A2.