Recombinant expression and identification of TP47 protein for serologic diagnosis of syphilis

Objective To express recombinant protein TP47 by genetic engineering technology and investigate the application in serologic diagnosis of syphilis.Methods TP47 gene was amplified by PCR from treponemal genomic DNA and cloned into vector pET28b.The recombinant plasmid pET28b-TP47 was transformed into E.coli BL21 for protein expression on IPTG induction.The expressed protein was purified by affinity chromatography and then identified by mass spectrometry.The reactivity of TP47 with syphilis patient sera was tested by Western blot.An ELISA based on TP47 antigen detection was developed and applied to test 30 TPPA positive and 75 TPPA negative sera.Results TP47 gene fragment about 1.1kb in length was amplified by PCR.The recombinant expression plasmid pET28b-TP47 was constructed correctly.Protein with molecular mass of 39 000 Mr was highly expressed and accumulated in inclusion body.The target protein was expressed in approximately 70% of total bacterial proteins.The purity of TP47 protein was about 95% after purification.The result of mass spectrometric analysis demonstrated that the expressed protein were TP47 protein which could react with syphilitic sera.The coincidence rate was 97% in TPPA positive sera and 97% in TPPA negative sera with the result of TP47-ELISA.Conclusions Recombinant TP47 antigen was successfully obtained by recombinant DNA technology and had strong reactivity with syphilitic sera.TP47 protein was an essential and feasible material for serologic diagnosis of syphilis.