摘要
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.
出处
《武汉大学学报(医学版)》
CAS
2016年第1期71-75,共5页
Medical Journal of Wuhan University
基金
国家自然科学基金(81300536
81172462)
湖北省自然科学基金(2013CFB331)
湖北省卫生厅青年人才项目(WJ2015Q025
QJX2012-22)
武汉科技大学绿色制造与节能减排科技研究中心开放基金(编号:C129)