抗弓形虫伴侣蛋白60多克隆抗体制备与鉴定

目的：制备、鉴定抗弓形虫伴侣蛋白60（TgCpn60）特异性多克隆抗体。方法：以弓形虫RH株cDNA为模板，PCR扩增编码TgCpn60的C末端168个氨基酸（TgCpn60C168AA）的507 bp序列片段，构建pGEX-4T-1-TgCpn60C168AA重组质粒。将鉴定正确的质粒转化入大肠埃希菌（E.coli ）BL21，并利用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，进而用Glutathione-SepharoseTM4B介质亲和层析法纯化重组蛋白后行SDS-PAGE电泳。以纯化的GST-TgCpn60C168AA蛋白作为免疫原免疫BALB/c小鼠，取小鼠血清进行纯化，获得特异性抗TgCpn60C168AA多克隆抗体。以该多克隆抗体为一抗，进行Western blot检测，验证其与虫体蛋白的反应情况。瞬时转染pFNR-RFP质粒至虫体后，采用间接免疫荧光法（IFA）检测TgCpn60与弓形虫顶质体外腔膜标志蛋白NADP+铁氧化还原蛋白还原酶（TgFNR）的共定位情况。结果：经PCR及测序结果证实重组质粒pGEX-4T-1-TgCpn60C168AA构建成功。SDS-PAGE电泳结果表明，GST-TgCpn60C168AA蛋白成功诱导表达。Western blot结果显示，制备的多克隆抗体既能特异性识别GST-TgCpn60C168AA重组蛋白，同时也能识别弓形虫内源性TgCpn60蛋白。IFA结果证实，TgCpn60蛋白与TgFNR蛋白存在共定位现象。结论：制备的抗GST-TgCpn60C168AA重组蛋白多克隆抗体能特异性识别弓形虫内源性TgCpn60蛋白，为进一步研究该蛋白功能提供了有利的工具。

Development and identification of polyclonal antibody against Toxoplasma gondii chaperonin 60(TgCpn60)

Objective: To develop and identify the specific polyclonal antibody against Toxoplasma gondii chaperonin 60 (TgCpn60). Methods: A 507 bp sequence fragment encoding the C-terminal 168 amino acids of TgCpn60 (TgCpn60C168AA) was amplified by PCR from T. gondii cDNA and then was cloned into prokaryotic expression vector pGEX-4T-1. The constructed plasmid pGEX-4T-1-TgCpn60C168AA was transformed into E.coli BL21 cells and induced by isopropyl-β-D-thinoglucoside (IPTG) for expression of the fusion protein tagging GST at its N-terminus, GST-TgCpn60C168AA. Recombinant fusion proteins were purified by Glutathione-SepharoseTM4B affinity chromatography. A polyclonal anti-serum was generated by immunizing BALB/c mice with purified GST-TgCpn60C168AA and was detected using Western blot assay. Furthermore, pFNR-RFP plasmid was transiently transfected into T. gondii RH strain, followed by IFA to detect the co-localization between TgCpn60 and T. gondii ferredoxin-NADP+ reductase (TgFNR), a marker protein for apicoplast lumen.Results: Successful construction of pGEX-4T-1-TgCpn60C168AA plasmid was confirmed by PCR amplification and sequencing. SDS-PAGE analysis showed that the recombinant GST-TgCpn60C168AA protein was expressed highly by IPTG induction. Western blot showed that both the recombinant GST-TgCpn60C168AA and native TgCpn60 from total parasite proteins could be recognized by the specific mouse anti-TgCpn60 polyclonal antibody. Moreover, the colocalization of TgCpn60 with the apicoplast marker FNR was demonstrated by IFA with the specific mouse polyclonal antibody. Conclusion: The obtained polyclonal antibody against the recombinant GST-TgCpn60C168AA can specifically react with the endogenous TgCpn60, which provides a favorable tool for further study into the function of TgCpn60.