弓形虫棒状体蛋白15的原核表达、纯化及多克隆抗体的制备  

Prokaryotic expression and purification of Toxoplasma gondii rhoptry protein 15(TgROP15)and production of polyclonal antibodies against TgROP15

作　　者：蒋鹏 王泽祥 车亮 王由森 孙晓林[1] JIANG Peng;WANG Zexiang;CHE Liang;WANG Yousen;SUN Xiaolin(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《甘肃农业大学学报》2023年第2期16-22,共7页Journal of Gansu Agricultural University

基　　金：甘肃省自然科学基金项目(21JR7RA823);家畜疫病病原生物学国家重点实验室开放基金项目(SKLVEB2019KFKT005)。

摘　　要：【目的】克隆弓形虫(Toxoplasma gondii)RH虫株速殖子的棒状体蛋白15(ROP15)基因,原核表达、纯化重组蛋白,并制备多克隆抗体。【方法】根据GenBank登录的弓形虫RH株ROP15(DQ096561.1)基因序列设计了1对引物,以提取的弓形虫RH虫株速殖子RNA为模板,通过RT-PCR获得该虫株的ROP15基因,将该基因连接至原核表达载体pGEX-4T-1上,获得重组质粒pGEX-4T-ROP15,测序结果与预期结果一致;转化BL21(DE3)感受态细胞,经IPTG诱导获得重组融合蛋白,运用亲和层析法纯化可溶性ROP15重组蛋白,SDS-PAGE对表达和纯化的目的蛋白进行分析。用纯化的ROP15重组蛋白免疫新西兰大白兔,制备多克隆抗体,运用Western blot分析特异性。【结果】成功克隆了弓形虫RH虫株速殖子ROP15基因,大小为924 bp;菌液PCR和酶切鉴定表明重组质粒pGEX-4T-ROP15构建成功,获得了约59 ku的可溶性融合蛋白表达产物;以纯化的可溶性ROP15重组表达蛋白为抗原制备兔源性抗血清,Western blot显示该多克隆抗体与ROP15重组蛋白和RH虫株速殖子总蛋白反应性良好。【结论】克隆出弓形虫RH虫株速殖子的棒状体蛋白15(ROP15)基因,利用原核表达系统获得了重组表达产物,纯化获得纯度较高的重组ROP15蛋白,制备的多克隆抗体与ROP15重组蛋白和RH虫株速殖子总蛋白反应性良好。【Objective】To clone the rhoptry protein 15(ROP15)gene of Toxoplasma gondii RH strain tachyzoites,to purify the recombinant proteins using a prokaryotic expression system,and to produce polyclonal antibodies.【Method】A pair of primers was designed according to the ROP15 gene sequence(DQ096561.1)of T.gondii RH strain available in GenBank,and the ROP15 gene of this strain was obtained by RT-PCR using the extracted tachyzoite RNA of Toxoplasma gondii RH strain as a template,followed by ligation of this gene into the prokaryotic vector pGEX-4T-1 to obtain the recombinant plasmid pGEX-4T-ROP15,which was sequenced,and the sequencing results were consistent with the expected results.BL21(DE3)E.coli competent cells were transformed and the recombinant fusion protein was obtained using IPTG.The soluble ROP15 recombinant protein was purified by affinity chromatography,and the expressed and purified target protein was analyzed by SDS-PAGE.Polyclonal antibodies were raised by immunizing New Zealand White rabbits with purified recombinant ROP15 protein and their specificity was analyzed by Western blot.【Result】The ROP15 gene of Toxoplasma gondii tachyzoites RH strain was successfully cloned,and its size was 924 bp.Bacterial liquid PCR and endonucleases digestion showed that the recombinant pGEX-4T-ROP15 was successfully constructed,and the soluble fusion protein expression product of about 59 Ku was obtained.Rabbit antiserum was raised using the purified soluble ROP15 recombinant expression protein as antigen,and Western blot showed that the polyclonal antibody could be specifically recognized by the recombinant ROP15 protein and total protein of RH strain tachyzoites.【Conclusion】The rhoptry protein 15(ROP15)gene of Toxoplasma gondii RH strain tachyzoites was cloned.The recombinant expression product was obtained by using the prokaryotic expression system,and the recombinant ROP15 protein was purified to high purity.The polyclonal antibody was specifically recognized by the recombinant ROP15 protein and by the t

关 键 词：弓形虫 棒状体 棒状体蛋白15 原核表达 纯化 多克隆抗体 

分 类 号：S855.9[农业科学—临床兽医学]