DnaJ类分子伴侣PBP基因的原核表达及兔抗PBP抗体的制备

Prokaryotic expression of DnaJ-homologous chaperon PBP and preparation of rabbit antibody against PBP

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。

AIM: To express DnaJ-homologous chaperon peripherin-binding protein(PBP) gene in E.coli and prepare the rabbit antibody against PBP. METHODS: The PBP cDNA was amplified from the human fetal brain tissue by RT-PCR. After confirmed by DNA sequencing, the PBP-cDNA was cloned into expression vector pET28a and then the PBP gene was expressed in E.coli under the IPTG induction. The expressed protein was purified through Ni-NTA affinity chromatography column. The rabbit antibody against PBP was prepared by immunizing two New Zealand white rabbits using the purified PBP as immunogen, The titer and specificity of the antisera were determined by Western blot. RESULTS: The 720 bp PBP gene was amplified, cloned, and expressed in E.coli. The expressed product existed in the bacterial inclusion body and the supernatant of the bacteria lysate. The purified PBP reached electrophoretic purity. The rabbit antibody against PBP was prepared and its titer was about 1∶1 600. Western blot analysis showed that the antibody could bind to the expressed PBP protein specifically. CONCLUSION: The PBP protein was expressed in E.coli and rabbit antibody against PBP was prepared successfully, which lays the foundation for further study on the structure and biological function of PBP.