Development and identification of polyclonal antibody against Toxoplasma gondii chaperonin 60(TgCpn60)
CAO Jiaxin1, CHENG Linyan2, LIU Shuxian3, TAN Feng3
1.School of Ophthalmology &Optometry, Wenzhou Medical University, Wenzhou 325035, China; 2.The First Clinical Medical College,Wenzhou Medical University, Wenzhou 325035, China; 3.School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou 325035, China
CAO Jiaxin,CHENG Linyan,LIU Shuxian, et al. Development and identification of polyclonal antibody against Toxoplasma gondii chaperonin 60(TgCpn60)[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2020, 50(11): 861-866.
Abstract：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.
