Development of an antigen detection based point-of-care test for the diagnosis of primary syphilis.

Abstract

Aim: To develop an antigen detection based, point-of-care test that will rapidly exclude syphilitic infection in patients presenting with genital ulcers. Materials and Method: T. pallidum subsp pallidum, Nichols strain, was propagated by intra-testicular inoculation of rabbits. T. pallidum DNA was obtained by suspending the testicular extract in ProbeTec lysis buffer followed by heating. Crude DNA was purified and concentrated. Specific primers were used for the amplification of the gene encoding the 31 kDa T. pallidum rare outer membrane porin protein (termed “Tromp1”). The amplified gene was cloned in frame with the pET100/D-TOPO vector that carries the N-terminal Xpress epitope and polyhistidine fusion tags. A screening PCR, restriction digest and DNA sequencing were used to confirm the presence of the tromp1 insert. Isolated plasmid DNA, pET100/D/tromp1 and the pET100/D/lacZ (positive control) were transformed into BL21 (DE3) pLysS E. coli cells for expression of recombinant Tromp1 and β-galactosidase as fusion proteins. SDS-PAGE and Western blot analysis were applied for detection of the recombinant proteins. Results: The gene encoding the 31 kDa Tromp protein was successfully cloned and sequenced. Multiple sequence alignment showed 100% homology between the cloned tromp1 gene sequence and its reference sequence. In addition, a screening PCR for transformation products and restriction digest of isolated plasmid DNA confirmed the presence of the tromp1 insert. Following gene expression, SDS-PAGE gel analysis showed no difference in the banding pattern between IPTG induced and uninduced lysates. The positive control however, showed a bright and distinct band at its expected size range of ~121 kDa. A Western blot and ELISA using specific antibodies to the N-terminal Xpress epitope fusion tag confirmed the absence of recombinant Tromp1 protein. Discussion and Conclusion: The results show that the tromp1 insert was successfully cloned and maintained up until the expression level. However expression of recombinant Tromp1 in BL21 (DE3) pLysS E. coli cells, for use as antigen in the serodiagnosis of primary syphilis was not achieved, despite several attempts to optimize gene expression. Expression of the positive control gene confirmed that growth and induction were properly performed.