[Study on ELISAs with rTpN17, rTpN47 and its fusion protein as antigens in the detection of serum samples from syphilis patients].
OBJECTIVE
To construct the prokaryotic expression systems of tpnl7 and tpn47 genes and tpn17-tpn47 fusion of Treponema pallidum, and to establish ELISAs based on rTpN17, rTpN47 and rTpN17-TpN47 as antigens to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosed syphilis.


METHODS
tpn17 and tpn47 genes were amplified and cloned by routine molecular biological methods. PCR with linking primers was used to construct artificial fusion gene tpn17-tpn47. The prokaryotic expression systems of the genes were then constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17, rTpN47 and rTpN17-TpN47. Ni-NTA affinity chromatography was applied to extract the three recombinant proteins, while Western blot was performed to determine their immunity. Using rTpN17, rTpN47 and rTpN17-TpN47 as the coated antigens, ELISAs (rTpN17-ELISA, rTpN47-ELISA and rTpN17-TpN47- ELISA) were established to detect serum samples from 200 healthy individuals, 25 RA patients, 17 SLE patients and 419 syphilis patients. Results of the ELISAs were compared to those with TRUST and TPHA.


RESULTS
The sequence similarities of the cloned tpnl7 and tpn47 genes and the constructed tpn17-tpn47 fusion gene were 100%, compared with the corresponding sequences in GenBank. The expression outputs of rTpN17, rTpN47 and rTpN17-TpN47 were 37.2%, 23.3% and 29.8% of the total bacterial proteins, respectively. Each of the three purified recombinant proteins showed a single fragment in gel after electrophoresis, and could take place remarkable conjugation reactions to the positive sera from syphilis patients. The detection results of rTpN17-ELISA, rTpN47-ELISA and rTpN17-TpN47-ELISA were negative for the serum samples from healthy individuals, RA and SLE patients, while presented 84.4%, 82.3% and 98.1% positive detection rates for the serum samples from syphilis patients. The positive detection rates of rTpN17-ELISA and rTpN47-ELISA were lower than that of TPHA (P<0.01), while the positive detection rate of rTpN17-TpN47-ELISA was similar to that of TPHA (P>0.05). All the positive detection rates from ELISA tests were higher than that of TRUST (71.4%).


CONCLUSIONS
rTpN17-ELISA, rTpN47-ELISA and especially rTpN17-TpN47-ELISA established in this study were of great hope as it was rapid, simple, convenient, safe, with high sensitivity and specificity for serological screening and detection of syphilis.
Sun
Ai-hua
AH
Zhejiang Medical College, Hangzhou, China.
Sun
Bai-li
BL
Yan
Jie
J
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005798:Genes, Bacterial; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012680:Sensitivity and Specificity; D013587:Syphilis / Q000175:diagnosis*; D013588:Syphilis Serodiagnosis / Q000379:methods*; D014210:Treponema pallidum / Q000235:genetics
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins