梅毒螺旋体重组蛋白rTp0608在梅毒血清学诊断中的初步应用
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摘要
目的建立以梅毒螺旋体重组蛋白Tp0608(rTp0608)为抗原的酶联免疫吸附试验(ELISA),进而评价其在梅毒血清学诊断中的价值。方法构建原核表达系统,镍离子金属鳌合亲和层析(Ni-NTA)纯化rTp0608,Western blotting鉴定目的蛋白并分析其免疫反应性。以该蛋白为包被抗原建立ELISA,检测梅毒患者及潜在交叉感染患者血清,并同梅毒螺旋体颗粒凝集实验(TPPA)进行比较。结果 Western blotting表明该蛋白能与梅毒阳性血清发生明显结合,但不能与健康人群及潜在交叉感染患者血清发生反应。rTp0608-ELISA阳性检测率为97.27%,与金标准的一致性为98.1%。结论 rTp0608-ELISA在检测梅毒患者血清时具有较高的灵敏性,且与金标准一致性较好。
Objective We established enzyme-linked immunosorbent assay(ELISA)based on recombinant protein Tp0608 to evaluate the effect on syphilis serological diagnosis. Methods The full length of tp0608 gene was amplified by polymerase chain reaction(PCR)and prokaryotic expression system was constructed. Sodium dodecylsulphate polyacrylamide gel electrophoresis(SDS-PAGE)was used to analyze the expression level of the recombinant protein. Nickel-nitrilotriacetic acid(Ni-NTA)affinity chromatography was utilized to purify recombinant protein Tp0608. Western blotting was performed to identify the protein and detect the reactivity between the recombinant antigen and the serum samples which were from syphilis patients and individuals with potential crossinfections. ELISA was established based on the antigen to determine the diagnostic potential using serum samples from 30 healthy individuals,5 leptospirosis patients,5 hepatitis B patients,5 hepatitis C patients and 110 syphilis patients. The κ coefficients were used to compare the concordance between gold standard and the Tp0608-based ELISA. Results The purified recombinant protein Tp0608 had strong conjugation reactions to the sera with positive antibody against Treponema pallidum,while there were no reactions observed between the recombinant antigen and serum samples from 5 healthy individuals,2 leptospirosis patients,2 hepatitis B patients,2 hepatitis C pa-tients and 10 syphilis patients. The positive detection rate of Tp0608-based ELISA was 97.27%,which was similar to that of gold standard. Moreover,the ELISA was in perfect agreement with the gold standard with a κ value of0.95. Conclusions ELISA based on recombinant antigen Tp0608 had high sensitivity in diagnosis of syphilis patients. The antigen can be a promising serodiagnostic candidate.
Objective We established enzyme-linked immunosorbent assay(ELISA)based on recombinant protein Tp0608 to evaluate the effect on syphilis serological diagnosis. Methods The full length of tp0608 gene was amplified by polymerase chain reaction(PCR)and prokaryotic expression system was constructed. Sodium dodecylsulphate polyacrylamide gel electrophoresis(SDS-PAGE)was used to analyze the expression level of the recombinant protein. Nickel-nitrilotriacetic acid(Ni-NTA)affinity chromatography was utilized to purify recombinant protein Tp0608. Western blotting was performed to identify the protein and detect the reactivity between the recombinant antigen and the serum samples which were from syphilis patients and individuals with potential crossinfections. ELISA was established based on the antigen to determine the diagnostic potential using serum samples from 30 healthy individuals,5 leptospirosis patients,5 hepatitis B patients,5 hepatitis C patients and 110 syphilis patients. The κ coefficients were used to compare the concordance between gold standard and the Tp0608-based ELISA. Results The purified recombinant protein Tp0608 had strong conjugation reactions to the sera with positive antibody against Treponema pallidum,while there were no reactions observed between the recombinant antigen and serum samples from 5 healthy individuals,2 leptospirosis patients,2 hepatitis B patients,2 hepatitis C pa-tients and 10 syphilis patients. The positive detection rate of Tp0608-based ELISA was 97.27%,which was similar to that of gold standard. Moreover,the ELISA was in perfect agreement with the gold standard with a κ value of0.95. Conclusions ELISA based on recombinant antigen Tp0608 had high sensitivity in diagnosis of syphilis patients. The antigen can be a promising serodiagnostic candidate.
引文
[1]WHO.WHO guidelines for the treatment of treponema pallidum(Syphilis)[Z/OL].http://apps.who.int/iris/bitstream/handle/10665/249572/9789241549806-eng.pdf;jsessionid=F0E16B8D7B9BEF01D7130347AD61C8C3?sequence=1
[2]TUCKER J D,COHEN M S.China's syphilis epidemic:epidemiology,proximate determinants of spread,and control responses[J].Curr Opin Infect Dis,2011,24(1):50-55.
[3]肖勇健,刘双全,谢亚锋,等.梅毒螺旋体大环内酯类抗生素耐药位点的监测[J].实用医学杂志,2017,33(15):2580-2583.
[4]MARRA C M,MAXWELL C L,DUNAWAY S B,et al.Cerebrospinal fluid treponema pallidum particle agglutination assay for neurosyphilis diagnosis[J].J Clin Microbiol,2017,55(6):1865-1870.
[5]WORKOWSKI K A,BOLAN G A.Sexually transmitted diseases treatment guidelines,2015[J].MMWR,2015,64(RR3):1-137.
[6]SERWIN A B,KOHL P K,CHODYNICKA B.The centenary of Wassermann reaction--the future of serological diagnosis of syphilis,up-to-date studies[J].Przegl Epidemiol,2005,59(3):633-640.
[7]VAN VOORHIS W C,BARRETT L K,LUKEHART S A,et al.Serodiagnosis of syphilis:antibodies to recombinant Tp0453,Tp92,and Gpd proteins are sensitive and specific indicators of infection by Treponema pallidum[J].J Clin Microbiol,2003,41(8):3668-3674.
[8]SMITH B C,SIMPSON Y,MORSHED M G,et al.New proteins for a new perspective on syphilis diagnosis[J].J Clin Microbiol,2013,51(1):105-111.
[9]JIANG C,ZHAO F,XIAO J,et al.Evaluation of the recombinant protein TpF1 of Treponema pallidum for serodiagnosis of syphilis[J].Clin Vaccine Immunol,2013,20(10):1563-1568.
[10]OSBAK K K,HOUSTON S,LITHGOW K V,et al.Characterizing the syphilis-causing treponema pallidum ssp.pallidum proteome using complementary mass spectrometry[J].PLoS Negl Trop Dis,2016,10(9):e0004988.
[11]LUKEHART S A,BAKER-ZANDER S A,LLOYD R M,et al.Characterization of lymphocyte responsiveness in early experimental syphilis.II.Nature of cellular infiltration and Treponema pallidum distribution in testicular lesions[J].J Immunol,1980,124(1):461-467.
[12]BRINKMAN M B,MCKEVITT M,MCLOUGHLIN M,et al.Reactivity of antibodies from syphilis patients to a protein array representing the Treponema pallidum proteome[J].J Clin Microbiol,2006,44(3):888-891.
[13]SCHMIDT B L,EDJLALIPOUR M,LUGER A.Comparative evaluation of nine different enzyme-linked immunosorbent assays for determination of antibodies against Treponema pallidum in patients with primary syphilis[J].J Clin Microbiol,2000,38(3):1279-1282.
[14]SAMBRI V,MARANGONI A,SIMONE M A,et al.Evaluation of recomWell Treponema,a novel recombinant antigen-based enzyme-linked immunosorbent assay for the diagnosis of syphilis[J].Clin Microbiol Infect,2001,7(4):200-205.
[15]JIANG C,XIAO J,XIE Y,et al.Evaluation of FlaB1,FlaB2,FlaB3,and Tp0463 of Treponema pallidum for serodiagnosis of syphilis[J].Diagn Microbiol Infect Dis,2016,84(2):105-111.
[16]XIE Y,XU M,WANG C,et al.Diagnostic value of recombinant Tp0821 protein in serodiagnosis for syphilis[J].Lett Appl Microbiol,2016,62(4):336-343.
[17]XU M,XIE Y,JIANG C,et al.A novel ELISA using a recombinant outer membrane protein,rTp0663,as the antigen for serological diagnosis of syphilis[J].Int J Infect Dis,2016,43:51-57.
[2]TUCKER J D,COHEN M S.China's syphilis epidemic:epidemiology,proximate determinants of spread,and control responses[J].Curr Opin Infect Dis,2011,24(1):50-55.
[3]肖勇健,刘双全,谢亚锋,等.梅毒螺旋体大环内酯类抗生素耐药位点的监测[J].实用医学杂志,2017,33(15):2580-2583.
[4]MARRA C M,MAXWELL C L,DUNAWAY S B,et al.Cerebrospinal fluid treponema pallidum particle agglutination assay for neurosyphilis diagnosis[J].J Clin Microbiol,2017,55(6):1865-1870.
[5]WORKOWSKI K A,BOLAN G A.Sexually transmitted diseases treatment guidelines,2015[J].MMWR,2015,64(RR3):1-137.
[6]SERWIN A B,KOHL P K,CHODYNICKA B.The centenary of Wassermann reaction--the future of serological diagnosis of syphilis,up-to-date studies[J].Przegl Epidemiol,2005,59(3):633-640.
[7]VAN VOORHIS W C,BARRETT L K,LUKEHART S A,et al.Serodiagnosis of syphilis:antibodies to recombinant Tp0453,Tp92,and Gpd proteins are sensitive and specific indicators of infection by Treponema pallidum[J].J Clin Microbiol,2003,41(8):3668-3674.
[8]SMITH B C,SIMPSON Y,MORSHED M G,et al.New proteins for a new perspective on syphilis diagnosis[J].J Clin Microbiol,2013,51(1):105-111.
[9]JIANG C,ZHAO F,XIAO J,et al.Evaluation of the recombinant protein TpF1 of Treponema pallidum for serodiagnosis of syphilis[J].Clin Vaccine Immunol,2013,20(10):1563-1568.
[10]OSBAK K K,HOUSTON S,LITHGOW K V,et al.Characterizing the syphilis-causing treponema pallidum ssp.pallidum proteome using complementary mass spectrometry[J].PLoS Negl Trop Dis,2016,10(9):e0004988.
[11]LUKEHART S A,BAKER-ZANDER S A,LLOYD R M,et al.Characterization of lymphocyte responsiveness in early experimental syphilis.II.Nature of cellular infiltration and Treponema pallidum distribution in testicular lesions[J].J Immunol,1980,124(1):461-467.
[12]BRINKMAN M B,MCKEVITT M,MCLOUGHLIN M,et al.Reactivity of antibodies from syphilis patients to a protein array representing the Treponema pallidum proteome[J].J Clin Microbiol,2006,44(3):888-891.
[13]SCHMIDT B L,EDJLALIPOUR M,LUGER A.Comparative evaluation of nine different enzyme-linked immunosorbent assays for determination of antibodies against Treponema pallidum in patients with primary syphilis[J].J Clin Microbiol,2000,38(3):1279-1282.
[14]SAMBRI V,MARANGONI A,SIMONE M A,et al.Evaluation of recomWell Treponema,a novel recombinant antigen-based enzyme-linked immunosorbent assay for the diagnosis of syphilis[J].Clin Microbiol Infect,2001,7(4):200-205.
[15]JIANG C,XIAO J,XIE Y,et al.Evaluation of FlaB1,FlaB2,FlaB3,and Tp0463 of Treponema pallidum for serodiagnosis of syphilis[J].Diagn Microbiol Infect Dis,2016,84(2):105-111.
[16]XIE Y,XU M,WANG C,et al.Diagnostic value of recombinant Tp0821 protein in serodiagnosis for syphilis[J].Lett Appl Microbiol,2016,62(4):336-343.
[17]XU M,XIE Y,JIANG C,et al.A novel ELISA using a recombinant outer membrane protein,rTp0663,as the antigen for serological diagnosis of syphilis[J].Int J Infect Dis,2016,43:51-57.