Objective: To clone tpn17 and tpn47 genes of Treponema pallidum and then construct their prokaryotic expression systems,to establish ELISAs based on rTpN17 and rTpN47 as antigens and to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosis of syphilis.
Methods: The whole length of tpn17 and tpn47 genes was amplified by PCR and then their prokaryotic expression systems were constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17 and rTpN47. Ni-NTA affinity chromatography was applied to extract rTpN17 and rTpN47, while Western blot was performed to determine the specific immunoreactivity of rTpN17 and rTpN47. By using rTpN17 and rTpN47 as the coated antigen, respectively, ELISAs (rTpN17-ELISA and rTpN47-ELISA) were established to detect serum samples from 200 healthy individuals, 25 RA patients, 17 SLE patients and 211 syphilis patients. The detection effects of the ELISAs were compared to those of TRUST and TPHA.
Result: The sequence similarity of the cloned tpn17 and tpn47 genes was 100 % compared with the corresponding sequences in GenBank. The expression outputs of rTpN17 and rTpN47 were approximately 37.2 % and 26.8 % of the total bacterial proteins, respectively. Both the extracted rTpN17 and rTpN47 could take place remarkable conjugation reactions to the sera with positive antibody against Treponema pallidum.The positive detection rate of TPHA (99.1%) was the highest (P<0.001). The positive detection rates of rTpN17-ELISA (85.3 %) and rTpN47-ELISA (84.3 %) were similar (P>0.05). The positive detection rates of TRUST (72.5 %) was lower than that of rTpN17-ELISA (P=0.001) but similar to that of rTpN47-ELISA (P=0.014). The detection results of all the serum samples from healthy individuals, RA patients and SLE patients were negative, whereas 7.1 % (3/42) of the samples from RA or SLE patients were positive.
Conclusion: rTpN17 and rTpN47 are still maintaining their original immunoreactivity. The ELISAs using rTpN17 or rTpN47 as the antigen are rapid, simple and convenient, higher sensitivity and specificity methods for serological screening and detection of syphilis.
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[Recombinant expression of the fusion antigen based on Treponema pallidum TpN17 and TpN47 epitope peptides and establishment and application of the associated ELISA].
Sheng Wu Gong Cheng Xue Bao
August 2009
Department of Basic Medicine, Zhejiang Medical College, Hangzhou 310053, China.
Using recombinant TpNs proteins of Treponema pallidum as antigens, ELISAs are proved to be of higher sensitivity and specificity. However, they can be further increased by using multiple TpNs antigens. According to the epitope analysis, we firstly used linking primers PCRs to obtain an artificial fusion gene segment tpE17-47 containing epitopes of both TpN17 and TpN47.
View Article and Find Full Text PDFSensitive and specific ELISA coated by TpN15-TpN17-TpN47 fusion protein for detection of antibodies to Treponema pallidum.
Clin Chem Lab Med
August 2009
Faculty of Basic Medicine, Zhejiang Medical College, Zhejiang Hangzhou, PR China.
Background: We constructed an artificial fusion gene tpN15-17-47 and then used the prokaryotic expression fusion protein rTpN15-17-47 as the coated antigen to establish a new enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of syphilis.
Methods: tpN15, tpN17, and tpN47 genes were amplified separately by polymerase chain reaction (PCR) and then assembled into a fusion gene coding a trigeminy protein antigen by primer linking PCR. The target recombinant protein antigens rTpN15, rTpN17, rTpN-47, and rTpN15-17-47 were expressed and then purified antigens were immobilized on the surface of microplate wells for detecting Treponema pallidum-specific antibodies by ELISAs.
[Study on ELISAs with rTpN17, rTpN47 and its fusion protein as antigens in the detection of serum samples from syphilis patients].
Zhonghua Liu Xing Bing Xue Za Zhi
October 2007
Zhejiang Medical College, Hangzhou, China.
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.
[Comparison of serological detection effects of ELISA using rTpN17 or rTpN47 of Treponema pallidum as antigen with that of TPHA and TRUST].
Zhejiang Da Xue Xue Bao Yi Xue Ban
January 2008
Zhejiang Medical College, Hangzhou 310053, China.
Objective: To clone tpn17 and tpn47 genes of Treponema pallidum and then construct their prokaryotic expression systems,to establish ELISAs based on rTpN17 and rTpN47 as antigens and to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosis of syphilis.
Methods: The whole length of tpn17 and tpn47 genes was amplified by PCR and then their prokaryotic expression systems were constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17 and rTpN47.
Peptide-protein microarrays for the simultaneous detection of pathogen infections.
Bioconjug Chem
November 2004
UMR CNRS 8527, Biological Institute of Lille, 1 Rue du Pr Calmette, 59021 Lille, France.
We describe novel peptide-protein microarrays, which were fabricated using semicarbazide glass slides that permitted the immobilization of glyoxylyl peptides by site-specific ligation and the immobilization of proteins by physisorption. The arrays permitted the simultaneous serodetection of antibodies directed against hepatitis C virus (HCV core p21 15-45 peptide, NS4 1925-1947 peptide, core, NS3, NS4, and mixture of core, NS3, NS4, and NS5 antigens), hepatitis B virus (HBc, HBe, and HBs), human immunodeficiency virus (Gp41 and Gp120 for HIV-I and Gp36 for HIV-II), Epstein-Barr virus (VCAp18 153-176 peptide), and syphilis (rTpN47 and rTpN17) antigens using an immunofluorescence assay. Peptide-protein microarrays displayed high signal-to-noise ratios, sensitivities, and specificities for the detection of antibodies as revealed by the analysis of a collection of human sera referenced against these five pathogens.
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