猪囊尾蚴排泄分泌抗原LRRC15蛋白的原核表达及多克隆抗体制备

Prokaryotic expression and polyclonal antibody preparation of LRRC15 protein, an excretory-secretory antigen of Cysticercus cellulosae

目的构建猪带绦虫重组质粒pET30a-富含亮氨酸重复序列(LRR)结构域15(LRRC15),原核表达纯化的LRRC15重组蛋白,制备兔多克隆抗体。方法采用全基因合成方法,获得猪带绦虫LRRC15蛋白编码基因;将其克隆至pET30a载体,构建重组质粒pET30a-LRRC15,并进行双酶切PCR鉴定;将重组质粒转化至大肠埃希菌BL21(DE3)感受态细胞,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达LRRC15重组蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析鉴定表达产物;用Ni-IDA亲和层析柱纯化LRRC15重组蛋白,SDS-PAGE进行分析鉴定;蛋白质印迹法(Western blot,WB)鉴定纯化重组蛋白特异性;用纯化的LRRC15重组蛋白免疫新西兰兔,制备LRRC15多克隆抗体,用间接酶联免疫吸附试验(ELISA)测定纯化多克隆抗体的效价。结果经PCR鉴定,扩增出长度为1506 bp的条带,与LRRC15基因相符;经SDS-PAGE和WB鉴定,获得相对分子质量(Mr)约为55.36×10^(3)的LRRC15目的蛋白;用LRRC15重组蛋白免疫新西兰兔,获得LRRC15多克隆抗体,其效价为1∶1587200。结论成功构建重组质粒pET30a-LRRC15,制备出猪带绦虫LRRC15重组蛋白,并获得了高纯度、高效价的LRRC15重组蛋白兔抗多克隆抗体。

Objective To construct a recombinant plasmid pET30a-leucine-rich repeat(LRR)containing 15(LRRC15)of Taenia solium,prokaryotically express and purify the LRRC15 recombinant protein,and prepare a rabbit polyclonal antibody.Methods The LRRC15 protein encoding gene of Taenia solium was obtained by whole gene synthesis;it was cloned into pET30a vector,and the recombinant plasmid pET30a-LRRC15 was constructed and identified by double-enzyme PCR;the recombinant plasmid was transformed into competent cells of Escherichia coli BL21(DE3),and the recombinant protein LRRC15 was induced to express by isopropyl-beta-D-thiogalactopyranoside(IPTG),the expression product was analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE);the LRRC15 recombinant protein was purified by Ni-IDA affinity columns,the purified recombinant protein was analyzed and identified by SDS-PAGE,and the specificity of the purified recombinant protein was identified by Western blot(WB);the New Zealand rabbits were immunized with purified LRRC15 recombinant protein to prepare polyclonal antibodies against LRRC15,and the potency of the purified polyclonal antibody was determined by indirect enzyme-linked immunosorbent assay(ELISA).Results After PCR identification,a band with a length of 1506 bp was amplified,which was consistent with the LRRC15 gene;after SDS-PAGE and WB identification,the LRRC15 target protein with a relative molecular mass(Mr)of about 55.36×10^(3) was obtained;after immunizing New Zealand rabbits with purified LRRC15 recombinant protein,a polyclonal antibody against LRRC15 was obtained,and its potency was 1∶1587200.Conclusion The recombinant plasmid pET30a-LRRC15 is successfully constructed,the LRRC15 recombinant protein of Taenia solium is prepared,and a high purity and high potency rabbit anti polyclonal antibody against LRRC15 recombinant protein is obtained.