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目的构建人Betatrophin基因(h-Betatrophin)原核表达载体,诱导Betatrophin重组蛋白表达及纯化,为制备Betatrophin蛋白多克隆抗体及其功能研究打下基础。方法体外克隆Betatrophin cDNA序列,酶切后连接到PET32a(+)原核表达载体上,构建重组载体h-Betatrophin-PET 32a,然后转入大肠杆菌BL 21(DE 3),用异丙基硫代半乳糖苷(IPTG)诱导蛋白表达,SDS-PAGE-考马斯亮蓝染色及Western blotting检测蛋白表达情况,然后用Ni-NTA His·BindResin纯化目的蛋白。结果成功构建Betatrophin基因原核表达载体,诱导表达Betatrophin重组蛋白表观分子量正确,经纯化后可以得到纯度较高的Betatrophin重组蛋白。结论重组载体h-Betatrophin-PET 32a转入大肠杆菌BL 21后通过诱导及纯化可获取到纯度很高的Betatrophin重组蛋白,为后期研究Betatrophin基因功能奠定基础。
Objective To construct the prokaryotic expression vector of human betatrophin gene (h-Betatrophin) and induce the expression and purification of Betatrophin recombinant protein, so as to lay the foundation for the preparation of polyclonal antibody against betatrophin protein and its function. Methods The cDNA sequence of Betatrophin was cloned in vitro and ligated into PET32a (+) prokaryotic expression vector. The recombinant vector h-Betatrophin-PET 32a was constructed and transfected into E. coli BL21 (DE3) The protein expression was induced by IPTG, SDS-PAGE-Coomassie brilliant blue staining and Western blotting. The protein was purified by Ni-NTA His · BindResin. Results The prokaryotic expression vector of Betatrophin gene was successfully constructed and the apparent molecular weight of Betatrophin recombinant protein was induced to be correct. After purification, Betatrophin recombinant protein with high purity could be obtained. Conclusion The highly purified Betatrophin recombinant protein can be obtained by induction and purification of the recombinant vector h-Betatrophin-PET 32a into E.coli BL21, which lays the foundation for the later study of betatrophin gene function.