人血清反应因子基因的原核表达及鸡卵黄多克隆抗体的制备

Prokaryotic expression of serum response factor and preparation of egg yolk antibody against serum response factor

目的在原核系统中表达人血清反应因子(serumresponsefactor,SRF)基因,制备鸡卵黄抗SRF抗体并鉴定其特性。方法构建SRF表达载体PGEX-T-2-SRF,并在大肠杆菌中以异丙基-β-D-硫代半乳糖苷(isopropy-β-D-thiogalactoside,IPTG)诱导表达。表达产物经谷胱甘肽-S转移酶(glutathione-S-transferase,GST)亲和层析柱纯化后,用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodiumdodecylsulphatepolyacrylamidegelelectrophoresis,SDS-PAGE)进行鉴定。用纯化的SRF免疫海蓝白母鸡,制备鸡抗SRF抗体。抗体的效价及特异性用蛋白质印迹技术进行测定和分析,免疫荧光鉴定其在心肌细胞中的定位。结果构建的重组质粒PGEX-4T-2-SRF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液中,纯化的SRF经SDS-PAGE鉴定呈单一条带。以纯化的SRF免疫制备了鸡抗SRF抗体。蛋白质印迹技术结果表明,该抗体可与原核表达的SRF特异性结合,抗体效价为1∶5000。免疫荧光证实该抗体可以和心肌细胞的细胞核特异性结合。结论在原核细胞中表达了SRF制备的鸡抗SRF的抗体对SRF能进行特异性结合。

Objectives To express serum response factor(SRF) gene in E.coli and prepare the egg yolk antibody against SRF.Methods After confirmed by DNA sequencing,the SRF cDNA was cloned into expression vector pGEX-4T-2 and then the SRF gene was expressed in E.coli under the IPTG in duction.The expressed protein was purified through GST affinity chromatography column.The egg yolk antibody against SRF was prepared by immunizing two New Zealand white hens with the purified SRF as immunogen,The titer and specificity of the antisera were determined by Western blot and immunofluorescence.Results The SRF gene was cloned and expressed in E.coli.The expressed product existed in the bacterial inclusion body and the supernatant of the bacteria lysate.The purified SRF reached electrophoretic purity.The egg yolk antibody against SRF was prepared and its titer was about 1∶5000.Western blot analysis showed that the antibody could bind to the expressed SRF protein specifically.Conclusions The SRF protein was expressed in E.coli and the egg yolk antibody against SRF was prepared successfully,which lays the foundation for further study on the structure and biological function of SRF.