弓形虫新的表面抗原SRS20A的鉴定及特征化

Identifi cation and Characterization of SAG1-Related-Sequence 20A Antigen from Toxoplasma gondii

克隆弓形虫上海SHR株SRS20A基因,构建原核表达体系,通过诱导表达和蛋白纯化,免疫BALB/c小鼠制备多克隆抗体,对蛋白进行特征化鉴定。采用PCR技术扩增弓形虫SRS20A基因,克隆至原核表达载体pET-30a(+)中,转化大肠埃希菌(E.coli),IPTG诱导目的蛋白表达,采用镍亲和层析法获得纯化蛋白并免疫BALB/c小鼠制备多克隆抗体,使用弓形虫阳性血清检测SRS20A蛋白的反应原性,应用纯化的rSRS20A蛋白通过ELISA方法对猪临床样品进行检测,利用Western blot技术检测多克隆抗体与弓形虫天然蛋白的反应性,通过间接免疫荧光观察SRS20A蛋白在虫体内的定位情况。成功从弓形虫基因组中扩增出SRS20A目的基因,构建了pET-30a-SRS20A重组质粒,利用小鼠感染弓形虫的阳性血清可以特异性识别重组蛋白rSRS20A,ELISA试验表明rSRS20A可以与猪临床血清特异性反应,证明SRS20A具有良好的反应原性。制备并获得了抗SRS20A重组蛋白的多克隆抗体,通过Western blot可检测出弓形虫天然蛋白SRS20A的特异性条带。通过间接免疫荧光方法鉴定SRS20A蛋白定位在弓形虫速殖子表面。通过分子生物学方法鉴定及特征化了一个新的弓形虫速殖子表面抗原SRS20A,为进一步深入研究弓形虫SRS20A功能奠定基础。

The major steps of the present study included cloning of the SAG1-related-sequence 20A(SRS20A)gene of Toxoplasma gondii Shanghai strain,construction of the prokaryotic expression system,purification of the recombinant protein and preparation of the polyclonal anti-SRS20A antibodies in BALB/c mice.The SRS20A gene of T.gondii was amplified by PCR and cloned into the prokaryotic expression vector pET-30a(+).Then the vector pET-30a(+)was transformed into E.coli for protein expression with induction of IPTG.The expressed rSRS20A protein was purified using nickel affinity chromatography and the purified protein was used to immunize BALB/c mice.Subsequently,Western blot was used to detect the reactivity of polyclonal antibodies with T.gondii native protein and rSRS20A protein was used to detect pig clinical samples by ELISA method.The localization of SRS20A protein inT.gondii was examined with indirect immunofluorescence assay using the positive murine serum.The results showed that the target SRS20A gene was successfully amplified from T.gondii genome and the recombinant plasmid pET-30a-SRS20A was constructed.The recombinant protein rSRS20A reacted with the positive serum of mice infected with T.gondii.The recombinant protein rSRS20A specifically reacted with the clinical serum samples of pigs,which proved its good reactivity.Polyclonal antibodies against the recombinant protein rSRS20A reacted with the natural SRS20A protein of T.gondii in Western blot.Moreover,the location of SRS20A protein on the surface of T.gondii tachyzoites was demonstrated in indirect immunofluorescence.In summary,a new surface antigen SRS20A of T.gondii tachyzoites was identified and characterized in the present study,which provided a reference for further research of the function of T.gondiiSRS20A.