目的 制备刚地弓形虫免疫球蛋白重链结合蛋白（TgBip）和真核翻译起始因子2α（TgeIF2α）多克隆抗体，并评价其特异性。方法 利用生物信息学方法设计TgBip和TgeIF2α基因的特异性引物，以刚地弓形虫RH株速殖子cDNA为模板，PCR扩增目的基因，采用无缝克隆技术，构建pColdⅢ-His-TgBip和pColdⅢ-His-TgeIF2α重组质粒，转化至大肠埃希菌BL21感受态细胞；异丙基-β-D-硫代半乳糖苷（IPTG）诱导蛋白表达后，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹（Western blotting）分析重组蛋白表达情况，采用Ni-NTA亲和层析纯化蛋白。以纯化后的TgBip和TgeIF2α蛋白为抗原免疫新西兰大白兔，制备特异性抗TgBip和TgeIF2α多克隆抗体。通过增强化学发光法分析多抗对内源性TgBip和TgeIF2α蛋白的识别情况，间接免疫荧光实验（IFA）检测TgBip蛋白在内质网应激过程中的变化，Western blotting检测内质网应激过程中TgeIF2α磷酸化水平的变化。结果 PCR结果显示，TgBip、TgeIF2α基因特异性扩增片段大小分别为1 920、1 044 bp，与预期片段大小相符。pColdⅢ-His-TgBip和pColdⅢ-His-TgeIF2α重组质粒构建成功。SDS-PAGE和Western Blotting结果表明，TgBip和TgeIF2α蛋白在BL21感受态细胞中高效表达，相对分子质量（M_r）约71 000和40 000，与理论值接近。Western blotting结果表明，制备的抗体可特异性识别弓形虫内源性TgBip和TgeIF2α蛋白，与宿主细胞蛋白无交叉反应。IFA结果表明，TgBip分布于弓形虫内质网，在诱导内质网应激前，TgBip分布在虫体细胞核周围；在内质网应激后，TgBip分布弥散。Western blotting结果表明，诱导内质网应激后磷酸化TgeIF2α相对表达水平为2.199 ± 0.376，高于诱导前的1.217 ± 0.099（t = 4.379，P < 0.05）。结论 制备的特异性抗TgBip和TgeIF2α多克隆抗体可用于检测弓形虫内质网结构与应激水平。

Objective To prepare polyclonal antibodies against Toxoplasma gondii immunoglobulin heavy chain-binding protein (TgBip) and eukaryotic translation initiation factor 2α (TgeIF2α), and to evaluate their specificity. Methods Specific primers for amplification of TgBip and TgeIF2α were designed using bioinformatics methods. Target genes were amplified using PCR assay with cDNA from tachyzoites of T. gondii RH strain as a template. The recombinant plasmids pColdⅢ-His-TgBip and pColdⅢ-His-TgeIF2α were constructed using in-fusion cloning, transformed into Escherichia coli BL21 competent cells. Following induction of protein expression with isopropyl-β-D-thiogalactoside (IPTG), the expression of recombinant proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting assay, and proteins were purified using Ni-NTA affinity chromatography. New Zealand white rabbits were immunized with purified TgBip and TgeIF2α proteins as antigens to prepare specific anti-TgBip and anti-TgeIF2α polyclonal antibodies. The recognition of endogenous TgBip and TgeIF2α proteins by polyclonal antibodies was analyzed using enhanced chemiluminescence assay. Changes in TgBip protein expression were examined using indirect immunofluorescence assay (IFA) during the process of endoplasmic reticulum stress, and changes in TgeIF2α phosphorylation levels were detected using Western blotting assay during the process of endoplasmic reticulum stress. Results PCR assay showed that the specific amplification fragments of TgBip and TgeIF2α were 1 920 and 1 044 bp in size, respectively, which were consistent with expected fragment sizes. The recombinant plasmids pColdⅢ-His-TgBip and pColdⅢ-His-TgeIF2α were successfully constructed. SDS-PAGE and Western blotting assay showed high expression of TgBip and TgeIF2α proteins in BL21 competent cells, with relative molecular masses (M_r) of approximately 71 000 and 40 000, which approached to the theoretical values. Western blotting assay showed that the prepared antibodies would specifically recognize endogenous T. gondii TgBip and TgeIF2α proteins, with no cross-reactions to host cells. IFA showed that TgBip was distributed in T. gondii endoplasmic reticulum, and TgBip was distributed around the cell nucleus prior to induction of endoplasmic reticulum stress and dispersed following induction of endoplasmic reticulum stress. Western blotting assay determined higher relative expression of phosphorylated TgeIF2α following induction of endoplasmic reticulum stress than prior to induction of endoplasmic reticulum stress [(2.199 ± 0.376) vs. (1.217 ± 0.099); t = 4.379, P < 0.05]. Conclusion The prepared specific polyclonal antibodies against TgBip and TgeIF2α may be used to detect the endoplasmic reticulum structure and stress level of T. gondii.