呼吸道合胞病毒F1片段膜外区的原核表达及其多克隆抗体制备  

Prokaryotic Expression of Extracellular Domain of Respiratory Syncytial Virus F1 Fragment and Preparation of Polyclonal Antibody

作　　者：张乐 巩新 刘波 吴军 ZHANG Le;GONG Xin;LIU Bo;WU Jun(Beijing Institute of Biotechnology,Beijing 100071,China)

机构地区：[1]军事医学研究院生物工程研究所,北京100071

出　　处：《生物技术通讯》2018年第4期496-500,共5页Letters in Biotechnology

基　　金：国家自然科学基金(81773619;81673339)

摘　　要：目的:利用大肠杆菌表达人呼吸道合胞病毒(RSV)F蛋白的F1片段膜外区,制备其多克隆抗体,为RSV的相关研究提供检测抗体。方法:采用全基因合成方法合成RSV的F1片段膜外区基因,构建带有His-Tag的原核表达载体p ET28a-F1-His,转化大肠杆菌BL21(DE3)细胞,37℃、IPTG诱导表达,超声波破菌,离心收集包涵体,溶解后经镍离子亲和层析方法进行纯化。纯化的蛋白经SDS-PAGE回收后切胶研磨,免疫大耳白兔,制备RSV的F蛋白多抗血清,采用Western印迹检测血清特异性。结果:构建的p ET28a-F1-His重组质粒在大肠杆菌中表达目的蛋白,其相对分子质量约44×10~3,与理论值一致,免疫后获得的多克隆抗体可特异性识别RSV F蛋白。结论:制备了RSV F蛋白的兔源多克隆抗体,为进一步研究RSV F蛋白疫苗奠定了基础。Objective： To express the extracellular domain of the F1 fragment of human respiratory syncytial virus（RSV） F protein and prepare a polyclonal antibody against RSV F protein to be used as a detection antibody in RSV study. Methods： The extracellular domain of the F1 fi＇agment of RSV F protein was synthesized by wholegenome synthesis technique, and it was inserted into prokaryotic expression vector to construct pET28a-F1-His followed by transformation into E.coli BL21 （DE3） cells. After target protein expression was induced by IPTG at 37℃, the inclusion bodies were released by ultrasonic disruption and collected by centrifugation, and then purified by nickel ion affinity chromatography. The purified protein was subjected to SDS-PAGE, and the protein strip was cut and ground to immunize the rabbit. The RSV F protein polyclonal antiserum was prepared, and the serum specificity was detected by Western blotting. Results： The constructed pET28a-F1-His recombinant plasmid expressed the target protein in E.coli with a relative molecular mass about 44x10^3 consistented with the theoretical size. The polyclonal antibody we obtained could specifically recognize RSV F protein. Conclusion： The rabbit polyclonal antibody against RSV F protein has been prepared, which laid a foundation for further research of RSV F protein vaccine.

关 键 词：呼吸道合胞病毒 F蛋白 原核表达 多克隆抗体 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]