柯萨奇-腺病毒受体基因的原核表达和多克隆抗体制备

目的利用原核基因工程克隆表达柯萨奇-腺病毒受体(CAR)胞外段,并制备多克隆抗体。方法从HeLa细胞中抽提总RNA,通过RT-PCR获得编码CAR胞外段的cDNA,与pQE31质粒连接,构建表达型重组质粒,测序证实后诱导表达,经Ni-NTA柱亲和纯化后,免疫新西兰兔,ELISA检测抗血清滴度。结果重组质粒转化的大肠杆菌M15经IPTG诱导表达及亲和纯化后得到滴度较高的融合蛋白,SDS-PAGE和Western blot显示其分子量约为26 kd,具有特异的抗原性,主要以包涵体形式存在于菌体中。纯化的融合蛋白免疫新西兰兔后产生的抗体,经ELISA检测抗体滴度>1:32 000。结论CAR胞外段基因的克隆表达和多克隆抗体的制备,为进一步研究该受体在以腺病毒载体为基础的基因治疗中的作用及柯萨奇病毒的致病机制奠定了基础。

Cloning and expression of human Coxsackie-adenovirus receptor gene and production of anti-CAR polyclonal antibody

Objective To construct expression plasmid encoding the extracellular domain of human CAR gene by prokaryotic genetic engineering. Methods Total RNA was isolated from the HeLa cells, and the cDNA encoding for the extracellular domain of CAR was obtained by RT-PCR and was cloned into pQE31 vector to construct recombinant expression plasmid. Fusion protein was induced by IPTG after verifying by sequencing and was purified by metal che-late affinity chromatography on Ni-NTA-sepharose. Purified protein was employed for immunization of New Zealand white rabbits. The anti-serum was evaluated by ELISA. Results Fusion protein was expressed in transformed host strain M15 after being induced by IPTG. The apparent molecular weight (26 kd) and immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot,and the expression product stayed mainly in precipitation as inclusion body in insoluble form. The rabbit anti-serum with high effectiveness ( titer > 1: 32 000) was produced and evaluated by ELISA. Conclusion The cloning, expression and purification of human CAR protein and the successful production of anti-CAR polyclonal antibody lay foundation for further study on the pathogenesis of CAR.