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

Objective:To express the outer membrane protein of Toxoplasma gondii (P30) in E.coli andpurify it, then analyse its immunoreactivity and immunogenicity for diagnosis oftoxoplasmosis.Methods:1. Expression, purification and renaturation of P30 protein DNA wasextracted from Toxoplasma gondii cultured in mouse macrophages as template. Theimmuno-dominant epitope of P30 gene was analysised by computer, then the P30gene was amplified from T. gondii complete genome by polymerase chain reactions(PCR). The PCR product was directly cloned into pUCm-T Vector. After beingidentified by enzymes cleavage analysis and PCR, the targe gene was subcloned intothe expression vector pET28b(+) to generate recombinant plasmid pET28b(+)/P30.The recombinant plasmid was inducted into E. coli BL21(DE3). After induced byIPTG, the expressed production was analyzed by SDS-PAGE and Western-Blot. Thefusion protein was purified by Ni-NTA affinity chromatography, and the purifiedprotein was analyzed by SDS-PAGE and Western-Blot. The protein concentration wasdetermined bythe BCA protein assay kit. 2. The preparation of mouse serumanti-Toxoplasma gondii and the collection of positive human serum from clinicKunming mouse were immunized with Toxoplasma gondii after three cycles offreezing and thawing at -80℃refrigerator, the polyclonal antibodies to anti-P30 inmouse serum were detected by indirected ELISA. The human IgG-positive serum were collected from clinic after sieved by the imported diagnose kit. 3. The establishof ELISA method to diagnose toxoplasmosis After coating wells with renaturatedprotein P30, the positive serum and the negative serum were detected byindirected-ELISA and the results were compared to the imported diagnose kit.Results:The size of PCR amplification product was about 800bp. According to thesequence reported by GenBank, the sequence analysis result showed that the targetfragment were P30 gene. A fusion protein with molecular weight about 30 kDa wasattained after it was expressed in E. coli, and it was existed as insoluble inclusionbodies (IBs). After lysised by 8M Urea, the protein was purificated with Ni²⁺chelating HiTrap HP column.The purity of the protein was above 95ï¼….Western-Blotting result proved that the recombinant protein can specifically react withT. gondii positive serum. Each well of microtiter maxisorp plate was coated with5μg P30 protein, then the positive or negative serum were added to detected IgGtargeting to T. gondii. After compared to the imported diagnose kit, the sensitivitiesand the specificities of the diagnostic reagent was 93.3ï¼…(14/15) and 100ï¼…(15/15)repectively and the total concordance of their results was 96.7ï¼…(29/30).Conclusion:1. Attained high purity of renaturation P30 protein.2. The renaturate P30 protein showed excellent immunoreactivity and it wasrecognized by T. gondii specific human IgG . The results lay the foundation fordevelopment of quick diagnostic kit applying to detect T. gondii.