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目的:克隆人结合珠蛋白(haptoglobin,Hp)cDNA,并在大肠杆菌中表达和鉴定。方法:从Hela细胞中分离总RNA,采用RT-PCR方法获得人Hp cDNA,分别克隆至原核表达载体pET-32a和PGEX-4T-1,转化至大肠杆菌BL21,IPTG诱导表达,并进行SDS-PAGE及Western blot鉴定。结果:成功构建了高效原核表达质粒PET-32a-Hp和PGEX-4T1-Hp;Western印迹结果表明,经IPTG诱导,在大肠杆菌中表达了分子量约30 kD和37 kD的目的蛋白;表达产物经Ni2+-NTA离子交换树脂纯化,纯度>90%。结论:在E.coli成功表达和纯化了人Hp融合蛋白,为进一步开发人Hp诊断试剂打下基础。
Objective: To clone human haptoglobin (Hp) cDNA and express and identify it in Escherichia coli. METHODS: Human Hp cDNA was isolated from Hela cells by RT-PCR and cloned into prokaryotic expression vector pET-32a and PGEX-4T-1 respectively. The recombinant plasmid was transformed into E.coli BL21 and induced by IPTG. SDS- PAGE and Western blot identification. Results: The prokaryotic expression plasmids PET-32a-Hp and PGEX-4T1-Hp were constructed successfully. The results of Western blotting showed that the target proteins of about 30 kD and 37 kD were expressed in E. coli by IPTG induction. Ni2 + -NTA ion exchange resin purification, purity> 90%. Conclusion: Human Hp fusion protein was successfully expressed and purified in E. coli, which laid the foundation for further development of human Hp diagnostic reagent.