BC022687融合蛋白在大肠杆菌中的表达、纯化、抗体制备及特异性鉴定

Expression,Affinity,Antibody Generation,Specificity Identification and Purification of a Mouse BC022687 Fusion Protein in E.coli

目的:构建BC022687的原核表达载体,诱导该质粒在大肠杆菌中表达并纯化其表达的融合蛋白,制备抗体并进行特异性鉴定。方法:采用RT-PCR法从18-20g体重雄性小鼠睾丸组织中扩增BC022687cDNA,经TA克隆及亚克隆方法后定向连入原核表达质粒pET-28a,将重组表达质粒转化至大肠杆菌BL-21(DL3)上,经IPTG诱导表达,SDS-PAGE分析,用考马斯亮蓝染色及Western blot分析鉴定后,利用镍亲和层析法纯化表达融合蛋白,免疫新西兰兔,制备多克隆抗体,以诱导表达的融合蛋白为样本,Western blot实验检测抗体的特异性。结果:测序结果证实成功扩增出目的基因BC022687;酶切和测序结果证实pET-28a-BC022687原核表达载体构建成功;SDS-PAGE电泳显示表达出18.0kU的外源蛋白。Western blot检测结果显示,表达出的蛋白为6×His tag的融合蛋白,而且Ni-NTA亲和层析法纯化该重组蛋白成功;将纯化的融合蛋白免疫新西兰兔,制备获得了抗BC022687蛋白的多克隆抗体,鉴定实验显示抗体特异性好。结论:已成功构建、表达、纯化了BC022687融合蛋白,以及成功制备了其特异性抗体,为研究BC022687蛋白在精子发生中的作用奠定了基础。

Objective：To construct a prokaryotic expressing vector of the mouse gene Bc022687 gene,and to express fusion protein and purify the recombinant protein by affinity chromatography,and to generate the polyclonal antibody and identify its specificity.Methods：The N-terminal of Bc022687 cDNA was amplified by PCR from mouse testis cDNA,the PCR product was cloned into pCR 2.1TA vector,and after sequencing the TA-cloning plasmid,the connected cDNA wassubcloned into pET-28 a.Then the plasmid was transformed into BL-21（DL3）cells after IPTG induction,which was detected by coomassie brilliant blue staining and Western blot analysis,and fusion protein was purified by Ni 2＋-NTA affinity purification.New Zealand rabbits were immunized with the purified protein to generate the polyclonal antibody.Induced fusion protein was used as samples,the specificity of anti-sera was identified by coomassie brilliant blue staining and Western blot analysis.Results：DNA sequencing and restriction endonuclease digestion analysis indicated that the prokaryotic expression vector BC022687/pET28 ahad been constructed successfully.After the recombinant plasmid being transformed,SDS-PAGE and Western blot analysis verified the expression of His6-BC022687 18.0kD fusion protein.The purified fusion protein could also be detected by SDS-PAGE analysis.Polyclonal antibody against BC022687 protein was successfully generated,and Western blot analysis indicated that the antibody specifically recognized BC022687 protein,not other nonrelated proteins.Conclusion：The N-terminal of cDNA Bc022687 was successfully cloned,expressed and purified in the form of His6 fusion protein,which is helpful for further study,such as antibody preparation and its function in spermatogenesis.