弓形虫TgERP基因的原核表达及间接ELISA方法的建立

Prokaryotic Expression of TgERP Gene of Toxoplasma gondii and Development of an Indirect ELISA

为原核表达弓形虫的TgERP基因,参照GenBank中弓形虫GTl株的TgERP基因序列设计引物,通过PCR扩增分离的一株猫源弓形虫(TC株)的TgERP基因,将其克隆至原核表达载体pET-28a中,通过IPTG诱导表达蛋白,应用带His标签的重力柱纯化蛋白,采用BCA方法测定蛋白浓度,利用重组蛋白免疫Balb/c小鼠制备多克隆抗体,并对重组蛋白进行SDS-PAGE和Western blotting分析。结果成功表达了TgERP蛋白,且蛋白以上清形式表达,相对分子质量约16 ku。将纯化的重组蛋白pET-28-TgERP作为诊断抗原,初步建立了间接ELISA方法;利用间接ELISA、改良凝集试验(MAT)和间接血凝试验(IHA)3种方法分别检测472份羊、犬、猫血清样品,发现阳性率分别为1.0%、3.8%、3.4%,验证了建立的间接ELISA方法可区分动物感染弓形虫的途径。本研究为弓形虫病快速诊断方法的建立和疫苗的研发奠定了基础。

In order to express the TgERP gene of Toxoplasma gondii by prokaryotic expression system,primers were designed with reference to the TgERP gene sequence of Toxoplasma gondii GTl strain registered in GenBank.The TgERP gene of a cat-derived Toxoplasma gondii(TC strain)was amplified by PCR,then cloned into the prokaryotic expression vector pET-28a to induce and express protein by IPTG,the TgERP protein was purified by Gravitrap with His-tag,and its concentration was measured by BCA method.The Balb/c mice were immunized with the recombinant protein to prepare polyclonal antibodies,and TgERP protein was analyzed by SDS-PAGE and Western blotting.The results revealed that recombinant TgERP protein was successfully expressed in the supernatant,and its molecular weight was about 16 ku.An indirect ELISA(iELISA)was developed taking the purified recombinant protein pET-28-TgERP as a diagnostic antigen;472 serum samples from sheep,dogs and cats were detected respectively by iELISA,modified agglutination test(MAT)and indirect hemagglutination test(IHA),and the positive rates were 1.0%,3.8% and 3.4%,respectively,which verified that the established iELISA could be used to distinguish the pathways of animal infection with Toxoplasma gondii.A foundation was thus laid for future development of a rapid diagnostic method for toxoplasmosis and corresponding vaccines.