| Objective To clone and express the two different fragments of the major surface antigen P30 of Toxoplasma gondii(Tg), obtained and purified recombinant proteins were used as antigens to diagnose toxoplasmasis. Method According to P30 gene sequence of toxoplasma, two pairs of primers were designed and synthesized, which were used to amplify the two different fragments of P30 gene from the genomic DNA of RH strain of Tg with PCR, and then the corresponding T/A clones were constructed with the two fragments respectively. PCR amplification and enzyme digestion after identifying the two fragments, then the fragments were sequenced and subcloned into PMAL-p2 plasmid, then transformed into susceptible E.coli DH5a to express. The positive clones were selected from the ampicillin positive LB plate after identification by PCR and enzyme digestion. The expressed proteins induced with IPTG were identified by western blotting. The identified proteins were purified by affinity chromatography with Amylose Resin.The serum of rabbits infected with Toxoplasma gondii were collected for the corresponding antibody ELISA detection in various sera from crude and purified recombinant antigens. Evaluate the detection results.Result The corresponding T/A clones and pMAL-p2 sub-clones have been successfully constructed, and the 73 and 70ku two protein fragments of P30 were expressed successfully. The purified recombinant antigens were applied to detect the antibodies in sera of rabbits infected with Toxoplasma gondii. The results showed that the recombinant antigens were more sensitive and specific than the crude antigen. Conclusion The two protein fragments of the major surface antigen P30 of Tg were obtained. They were used to detect the antibodies in sera of rabbits infected with Tg successfully, which might be applied to immunodiagnosis of patients with Toxoplasmiasis. |
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