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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,将其克隆到原核表达载体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。
Objective To construct a prokaryotic expression vector for extracellular domain of human follicle stimulating hormone receptor (FSHR) and immunize mice with purified recombinant protein to prepare antiserum. Methods The extracellular domain of human FSHR was cloned by RT-PCR and cloned into prokaryotic expression vector pET32a (+). The target gene was expressed in E. coli through IPTG. The obtained inclusion body protein passed through Ni-NAT ion exchange column The recombinant protein was purified and identified by SDS-PAGE and Western blotting. Results The gene fragment of 1 047 bp was amplified by PCR. The sequencing confirmed that the cloned gene sequence was consistent with the FSHR sequence in GenBank. The engineered bacteria pET32 a (+) / FSHR extracellular domain induced by IPTG expression, SDS-PAGE showed a new protein expression band, Mr about 58 000, with the expected. Its expression form is insoluble inclusion body. The inclusion body protein is purified by Ni-NAT ion exchange column chromatography. The purified protein can be seen by SDS-PAGE analysis of a single band. The protein was used to immunize mice to prepare antiserum with the titer of 1:12 800. Western blot showed that the antibody could specifically bind to the target protein. Conclusion The human FSHR extracellular fusion protein and specific polyclonal antibody were obtained, which laid the foundation for further study of the function of FSHR protein.