Expression And Purification Of P30 Outer Membrane Recombinant Protein Of Toxoplasma Gondii And Evaluation Of Its Diagnostic Potential

Objective:To express the outer membrane protein of Toxoplasma gondii (P30) in E.coli and purify it , then analyse its immunoreactivity and immunogenicity for diagnosis of toxoplasmosis.Methods:1. Expression, purification and renaturation of P30 protein DNA wasextracted from Toxoplasma gondii cultured in mouse macrophages as template. The immuno-dominant epitope of P30 gene was analysised by computer, then the P30 gene was amplified from T. gondii complete genome by polymerase chain reactions (PCR). The PCR product was directly cloned into pUCm-T Vector. After being identified by enzymes cleavage analysis and PCR, the targe gene was subcloned into the expression vector pET28b(+) to generate recombinant plasmid pET28b(+)/P30. The recombinant plasmid was inducted into E. coli BL21(DE3). After induced by IPTG, the expressed production was analyzed by SDS-PAGE and Western-Blot. The fusion protein was purified by Ni-NTA affinity chromatography, and the purified protein was analyzed by SDS-PAGE and Western-Blot. The protein concentration was determined by the BCA protein assay kit. 2. The preparation of mouse serum anti-Toxoplasma gondii and the collection of positive human serum from clinic Kunming mouse were immunized with Toxoplasma gondii after three cycles of freezing and thawing at -80℃ refrigerator, the polyclonal antibodies to anti-P30 in mouse serum were detected by indirected ELISA. The human IgG-positive serum...