摘要
根据GenBank上查到的人胶原蛋白序列COL6A2(NM058175.2),利用Primer 5设计特异性引物,以吉林农业大学生物物理实验室构建的重组质粒pCMV-Sport6-COL6A2为模板进行目的基因的克隆,获得目的基因COL6A2,然后用双酶切的方法连接目的基因COL6A2和表达载体pET32a,将连接产物转化到大肠杆菌中构建原核表达载体pET32a-COL6A2。再将鉴定成功的重组质粒pET32a-COL6A2分别转化到大肠杆菌BL21、BL21(DE3)、BL21(BE3)plysS及Rosetta(DE3)中,采用1 mmol/L的异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达,经12%SDS-PAGE电泳分析并筛选出重组蛋白表达量最高的菌株。利用Ni Sepharose 6 FastFlow琼脂糖树脂亲和层析柱纯化重组蛋白。结果表明:获得了大小为570 bp的COL6A2片段,经测序鉴定序列正确;成功构建了pET32a-COL6A2原核表达载体并表达出约30 kD的目的蛋白;筛选出BL21(DE3)作为高效表达菌株,其重组蛋白表达量占菌体总蛋白的23.9%;经镍离子亲和层析纯化获得了纯度>90%的目的蛋白。
According to the sequence of COL6A2 gene（NM0581 75.2）from GenBank of NCBI,specific DNA primers were designed by primer 5 .Target gene COL6A2 was obtained by PCR from the recombi-nant plasmid of pCMV-Sport6-COL6A2 structured by laboratory,gene COL6A2 and pET32a vector were connected by restriction endonuclease digestion assay .The recombinant plasmids of pET32a-COL6A2 were transformed into E .coli BL21 ,BL21 （DE3）,BL21 （BE3）plysS and Rosetta（DE3）respectively and induced by IPTG .Then the best efficient strain was selected by SDS-PAGE assay .The recombinant protein was purified by Ni Sepharose 6 Fast Flow.The results show that cDNA sequence of COL6A2 was cloned correctly .The vector of pET32a-COL6A2 was constructed and the target protein about 30 kD was expressed successfully .E .coli BL21 （DE3）was screened as efficient expression strain and the expression level of target protein was 23.9 %.The target protein whose purity is more than 90%was obtained by Ni Sepharose 6 Fast Flow .The results lay foundation for the research of recombinant collagen activity .
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
2014年第1期51-55,65,共6页
Journal of Jilin Agricultural University
基金
吉林省自然科学基金项目(201115189)
关键词
胶原蛋白
重组载体
大肠杆菌
克隆
纯化
collagen
recombinant vector
Escherichia coli
cloning
purification
