Preparation And Application Of The Recombinant Antigen P22 Of Toxoplasma Gondii

Objective: To obtain highly sensitive and specific recombinant antigen P22 ,the major surface antigen of Toxoplasma gondii (Tg), by gene recombinant technique , which was applied to the diagnosis of the Tg infection.Method: The P22 gene expressed the Tg major surface antigen P22 was amplified from Tg RH strain genomic DNA. The above fragment was inserted into T/A vector and identified by PCR, enzyme digestion and gene sequencing. The positive clone identified was amplified. Recombinant plasmid was obtained by QIAprep Spin Miniprep Kit. P22 gene obtained by enzyme digestion was purified with the QIAquick Gel Extraction Kit. The purified gene and prokaryotic plasmid pET-32a were recombinated into recombinant plasmid with T4 DNA ligase. The recombinant plasmid identified by PCR, enzyme digestion and gene sequencing was converted into E.coli BL21, then induced by IPTG. After analyzed by SDS-PAGE and Western blot, the expression product was dissolved in urea and purified by affinity to NTA column. The purifiedprotein was washed and dialysed step by step to get rid of urea. The purified product was renatured by Oxidation/reduction GSH to obtain the recombinant antigen. Finally, the sera from the rabbits infected with Tg and various patients were detected comparatively with the Tg recombinant antigen P22 and crude antigen.Result: 1. The gene obtained from Tg genomic DNA was confirmed to be P22 gene. 2. Plasmid pET-32a/P22 was recombinated successfully. 3. The recombinant antigen P22 was obtained. 4. The recombinant antigen P22 has high sensitivity and specificity: its detective rate for Tg infected rabbits is as high as 96.7%, while has no any cross reactions with other none Tg infected animals and patients.Conclusion: The recombinant antigen P22 obtained successfully is highly sensitive and specific in the detection of Tg antibody.