Treponema Pallidum Expression, Purification And Identification Of Recombinant Tpn17 Antigen

Syphilis is a very harmful sexually transmitted disease of humans over the world that is caused by infection with the bacteria Treponema pallidum subsp. pallidum (T. pallidum). Serological test of syphilis has the advantages of high-speed, easily handling and low cost, and has been used in clinica extensively. However, the current serological testing assay of syphilis does not present the satisfied sensitivity and specificity. Enzyme-linked immunosorbent assay (ELISA) based on genetic engineering antigen instead of natural antigen would provide the solution to improve the sensitivity and specificity in serological examination. In this study, we produced a recombinant lipoprotein antigen designated TpN17 by genetic engineering technique. This protein could be used to as a key antigen in the development of ELISA diagnostic kit of syphilis.First of all, in this study, we cloned target gene encoding mature peptide TpN17 from Tp genome by using polymerase chain reaction (PCR) technique. Then, the gene was inserted into vector by restriction enzyme HindIII and EcoRI digestion. The construct was tansformed into the competent E.coli BL21 (DE3 ) for expression. The recombinants were screened by antibiotic Kanamycin plate, then it was confirmed by PCR, restriction enzyme digestion and DNA sequencing. Under the induction with 1.0mmol/L IPTG at 37℃ for 4 hours, the engineering strain BL21/pET-28b(+)/tpnl7 could produce large amount of recombinant protein. SDS-PAGE analysis indicated that the molecular weight of the recombinant protein was about 19kD, which is very close to the molecular weight of His-TpN17 fusion protein(18.7kD ). Scanning analysis indicated that the induced target protein (His-TpN17 fusion protein ) accounts for about 31% of the total bacterial proteins.Secondly, we found that His-TpN17 fusion protein was expressed primarily in the form of soluble supernatant intracellularly in E.coli. In the process of purification for His-TpN17 fusion protein, more soluble target protein could be obtained by using repeat freeze-thaw with lysozyme and washing bacteria cellular debris repeatedly. In addition, SDS-PAGE indicated that the molecular weight of recombinant protein purified by Ni²⁺ chelate chromatography was about 19kD and its purity reached above 95%. Western-blot further confirmed that the purified product was TpN17 recombinant antigen because it could react specifically with human secondary syphilitic sera. In conclusion, wesuccessfully cloned the gene of TpN17 lipoprotein and produced the recombinant in E.coli system. The purified recombinant of TpN17 protein could be used as a key antigen in the manufacturing of ELISA diagnostic kit of syphilis.