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目的:在原核系统中表达DnaJ类分子伴侣感光受体外周蛋白结合蛋白(PBP)基因,并制备兔抗PBP的抗体鉴定其特性。方法:应用RTPCR从人胎脑组织总RNA中扩增PBPcDNA。测序后将其克隆到表达载体pET28a中,并在大肠杆菌中以IPTG诱导表达。表达产物经NiNTA亲和层析柱纯化后,用SDSPAGE进行鉴定。以所获纯化的PBP免疫新西兰白兔,制备兔抗PBP抗体。抗体的效价及特异性用Westernblot进行测定和分析。结果:扩增和克隆出了720bp的PBP基因。构建的重组质粒pET28aPBP在大肠杆菌中得到高表达,诱导表达的蛋白存在于包涵体和细菌裂解上清中,纯化的PBP经SDSPAGE鉴定呈单一条带。以纯化的PBP免疫兔,制备了兔抗PBP抗体。Westernblot鉴定证实,该抗体可与原核表达的PBP特异性结合,抗体效价为1∶1600。结论:在原核细胞中表达了具有生物学活性的PBP,并以其为免疫原制备了兔抗PBP的抗体,为进一步研究PBP的结构与生物学活性奠定了基础。
OBJECTIVE: To express the peripheral binding protein (PBP) gene of DnaJ chaperone in prokaryotic system and to prepare the anti-PBP antibody. Methods: PBP cDNA was amplified from human fetal brain tissue by RTPCR. After sequencing, it was cloned into the expression vector pET28a and induced by IPTG in E. coli. The expressed product was purified by NiNTA affinity chromatography and identified by SDSPAGE. Rabbit anti-PBP antibodies were prepared by immunizing New Zealand white rabbits with purified PBP. The titer and specificity of the antibodies were determined and analyzed by Western blot. Results: The 720 bp PBP gene was amplified and cloned. The constructed recombinant plasmid pET28aPBP was highly expressed in E. coli. The induced protein was present in inclusion bodies and bacterial lysate supernatant. The purified PBP was identified as a single band by SDSPAGE. Rabbit anti-PBP antibodies were prepared by immunizing rabbits with purified PBP. Westernblot identification confirmed that the antibody specifically binds to prokaryotic expressed PBP with an antibody titer of 1: 1600. CONCLUSION: The biologically active PBP is expressed in prokaryotic cells and its anti-PBP antibody is prepared as an immunogen, which lays the foundation for further study on the structure and biological activity of PBP.