摘要
目的:克隆编码果蝇硒蛋白D-SelK结构基因并在大肠杆菌中表达,纯化后制备兔抗D-SelK的抗体。方法:利用PCR从pGM-T-D-SelK质粒中扩增D-SelK基因,构建重组表达质粒pGEX-6p-1-D-SelK,以重组质粒转化E.coli BL21(DE3),筛选阳性重组菌株,IPTG诱导目的蛋白表达,通过变性、电泳纯化D-SelK蛋白,SDS-PAGE和Westem blot方法鉴定目的蛋白的表达;以表达的D-SelK蛋白免疫家兔,制备抗D-SelK的多克隆抗体并进行效价及特异性鉴定。结果:扩增了D-SelK基因,克隆于表达载体pGEX-6p-1中,PCR筛选和酶切鉴定获得阳性克隆,测序证实正确。经诱导在大肠杆菌中表达出目的蛋白,纯化后免疫家兔,能有效地刺激特异性抗体的产生,抗血清的效价达到1∶51 200以上,且具有良好的特异性。结论:已成功构建D-SelK基因的原核表达载体,并在大肠杆菌中获得高效表达;制备出兔抗D-SelK抗体,效价及特异性均良好,这为进一步研究功能特异性的D-SelK提供了有效的手段。
AIM： To clone and encode Drosophila selenoprotein D-SelK structure gene,express it in E.coli efficiently,and after purification,to generate its antibody in rabbits.METHODS： D-SelK gene segment amplified from pGM-T-D-SelK plasmid by PCR was inserted into pGEX-6p-1 to construct recombinant plasmid pGEX-6p-1-D-SelK.The recombinant plasmid was transfected into E.coli BL21（DE3） to express the recombinant protein D-SelK in E.coli under IPTG induction.The protein was purified by denaturation and electrophoresis,and then identified by SDS-PAGE and Western blotting.Polyclonal antibody to D-SelK was obtained by immunizing rabbits with the protein.Quality and quantity of the antibody was examined.RESULTS： D-SelK gene segment was successfully inserted into pGEX-6p-1 and the positive clones of the recombinant plasmid was identified by PCR screening and restriction endonuclease analysis.The target protein was effectively expressed in E.coli by the IPTG induction.Through immunizing rabbits with the purified target protein,we obtained the specific antibodies to D-SelK,the titer of which was more than 1∶ 51 200.The polyclonal antibody had a good specificity to D-SelK.CONCLUSION： D-SelK recombinant protein and rabbit anti-D-SelK polyclonal antibody with high specificity were obtained,which provides good tools for further research on the functional characterization of D-SelK.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2012年第8期844-846,850,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助(30671176)
辽宁省教育厅资助(2009A310)
辽宁省科学技术计划项目(2010225036)
关键词
果蝇硒蛋白
D-SelK
原核表达
多克隆抗体
Drosophila selenoprotein
D-SelK
prokaryotic expression
polyclonal antibody
