FSHR胞外区基因片段的原核表达及多克隆抗体的制备

目的 构建人卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)胞外区的原核表达载体,并用纯化的重组蛋白免疫小鼠,制备抗血清.方法 采用RT-PCR克隆人FSHR胞外区,将其克降到原核表达载体pET32a(+),并通过IPTG诱导目的 基因在大肠杆菌中表达,所获得的包涵体蛋白通过Ni-NAT离子交换柱层析纯化重组蛋白,并用SDS-PAGE和蛋白印迹法鉴定.结果 PCR扩增出1 047 bp目的 基因片段,测序证实克隆的基因序列与GenBank中的FSHR序列相符.工程菌pET32 a(+)/FSHR胞外区经IPTG诱导表达后,SDS-PAGE显示有新生的蛋白表达条带,Mr约为58 000,与预期的一致.其表达形式为不溶性包涵体,此包涵体蛋白通过Ni-NAT离子交换柱层析纯化,纯化的蛋白质经SDS-PAGE分析可见单一条带.该蛋白免疫小鼠制备抗血清,抗体效价为1:12 800,Western blot检测证实该抗体能与目的 蛋白发生特异性结合.结论 获得了人FSHR胞外区融合蛋白及特异性多克隆抗体,为进一步研究FSHR蛋白的功能奠定了基础.

Cloning and expression of extracellular region of human FSHR in E.coli

Objective To construct a prokaryotic expression vector of extracellular region of human follicle stimulating hormone receptor(FSHR),purify this protein and prepare its antiserum.Methods Total RNA was isolated from human testis,while extracellular region gene of human FSHR was amplified with RT-PCR.The enzyme-digested target fragment was cloned into pET32 a(+) expression vector,and then transferred into E.coli BL21(DE3),in which extracellular region of human FSHR was induced to express by IPTG.After the incl...