关键词: 刚地弓形虫, 内质网应激相关蛋白, 免疫球蛋白重链结合蛋白, 真核翻译起始因子2α, 多克隆抗体

Abstract:

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.

Key words: Toxoplasma gondii, Endoplasmic reticulum stress-related protein, Immunoglobulin heavy chain-binding protein, Eukaryotic translation initiation factor 2α, Polyclonal antibody