新基因Betatrophin原核重组蛋白表达及纯化

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PET32 a(+)原核表达载体上,构建重组载体h-Betatrophin-PET 32 a,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·Bind(R)Resin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 32 a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.

Construction of Prokaryotic Expression Vector of New Gene Betatrophin and Its Recombition Protein Purification

Objective To construct the Prokaryotic expression vector of human betatrophin(h-betatrophin) gene and induce the expression of betatrophin recombinant protein,and purify target protein,which makes preparation of the production of Betatrophin protein polyclonal antibody and lies a foundation for research this gene's function.Methods Cloned Betatrophin cDNA sequence in vitro was inserted to PET 32 a (+) expression vector through double enzyme digestion and established the recombinant expression vector h-Betatrophin-PET 32 a.h-Betatrophin-PET 32 a vector was then transformed into E.coli BL 21 (DE 3).The expression of Betatrophin protein was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG) in BL 21,and confirmed by SDS-PAGE-commassie blue fast staining solution and western-blot analysis.In addition,the Betatrophin recombinant protein was purified by Ni-NTA His·Bind(R) Resin.Results The recombinant vector h-Betatrophin-PET 32 a was constructed.The epigenetic molecular weight of induced Betatrophin recombinant protein was correct,and high purity Betatrophin recombinant protein was successfully purified.Conclusion Recombinant expression vector h-Betatrophin-PET 32 a was transformed into BL 21 and could efficiently express h-Betatrophin recombinant protein.A large number of homogeneous h-Betatrophin proteins can be obtained by our purification methods.