摘要
目的 得到大量纯的βB2 -晶体蛋白,为研究其生物学特性及潜在的生理功能打下基础.方法 从SD大鼠晶状体中抽提总RNA ,用RT -PCR法克隆βB2 -晶体蛋白的cDNA ,装入原核表达载体pGEX - 4T - 1后用萘啶酮酸(IPTG)诱导表达;用GST纯化系统纯化并用蛋白水解酶对融合蛋白进行酶切.结果 βB2 -pGEX重组质粒经测序及限制性内切酶分析等鉴定,与预期结果一致;IPTG诱导后,蛋白质在大肠杆菌BL2 1中得到高效表达.纯化酶切后,经Westenblot方法验证,得到了高纯度的βB2 -晶体蛋白.结论 成功构建了βB2 -pGEX原核表达载体,高效表达了βB2-GST融合蛋白,经纯化和酶切得到了高纯度的βB2 -晶体蛋白,为进一步研究其生物学特性及潜在的生理功能打下基础.
Objective To obtain lots of pure βB 2-crystallin for the base of studying it's biologic characteristic and potential physical functions. Methods The cDNA of Rat lens was obtained by PR-PCR using the total RNA got from rat lens as moulding board, then cloned into pGEX-4T-1 vector and induced by IPTG. Purification and cutting by protease was done refer to the specification of the GST purification system. Results The constructed recombinant βB 2-pGEX plasmid had been identified by restriction endonuclease cutting and sequencing. The expression of the recombinant plasmid was identified by Western blot. Conclusions The recombinant βB 2-pGEX plasmid had been constructed and expressed correctly, and the purification was successful. The obtainng of pure βB 2-crystallin lays the further study of it's biologic characteristic and potential physical functions.
出处
《现代临床医学生物工程学杂志》
2005年第1期1-4,共4页
Journal of Modern Clinical Medical Bioengineering
基金
国家自然科学基金 (30 4 70 681 ) .
