Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Metho...Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.展开更多
Objectives： To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods： The immuno-dominant epitope of Tp0453 gene was amplif...Objectives： To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods： The immuno-dominant epitope of Tp0453 gene was amplified from the complete genome of T.pallidum by polymerase chain reactions （PCR）, subcloned into the expression vector Pqe32 to generate recombinant plasmid Pqe32/Tp0453, and was then expressed in E. coli M15. The fusion protein was purified with Ni-NTA affinity purification. Indirect ELISA was developed to detect human serum IgG antibody to T. pallidum. Results： The recombinant Tp0453 protein was successfully expressed and purified. The recombinant protein had a molecular weight of approximately 32KDa.Indirect ELISA to the recombinant protein was developed.Sixty control sera were tested by ELISA and yielded a sensitivity of 100% （30/30） and a specificity of 100% （30/30）. While testing for T. pallidum in human sera, the sensitivity and specificity of ELISA were 96.8% and 100%, respectively, when compared with TPPA test results. The concordance of results between the ELISA test and the TPPA test was 98.2%. Conclusion： The recombinant Tp0453 outer membrane protein elicited a strong immunoreaction to anti-T.pallidum IgG antibody and has great potential use in ELISA for the serodiagnosis of syphilis.展开更多
Objectives: To develop a multi-nested polymerase chain reaction in an assay to detect early Treponema pallidum and Haemophilius ducreyi DNA in the swabs of genital ulcers. Methods: Four pairs of outer and inner primer...Objectives: To develop a multi-nested polymerase chain reaction in an assay to detect early Treponema pallidum and Haemophilius ducreyi DNA in the swabs of genital ulcers. Methods: Four pairs of outer and inner primers, specific to the basic membrane protein gene of Treponema pallidum and to the 16s rRNA gene of H ducreyi were synthesized. The multi-nested PCR was developed and applied to detect Treponema pallidum and Haemophilus dicreyi in clinical swabs. Result: The two samples of standard strains of Haemophilus ducreyi and one Treponema pallidum were amplified and showed 309-bp rRNA gene of Haemophilus ducreyi and 506-bp DNA of Treponema palidum, respectively. Out of 51 samples of genital ulcer detected, 29 showed Treponema pallidum positive product and no Haemophilus ducreyi DNA was found. Conclusion: The multi-nested PCR for Treponema pallidum and Haemophilus ducreyi could be useful for early detection and distinguishing diagnosis between syphilis and chancroid.展开更多
Objective To assess the sensitivity, specificity, and feasibility of 4 recombinant Treponema pallidum antigenbased rapid tests in the diagnosis of syphilis. Methods A total of 970 outpatients were selected from the S...Objective To assess the sensitivity, specificity, and feasibility of 4 recombinant Treponema pallidum antigenbased rapid tests in the diagnosis of syphilis. Methods A total of 970 outpatients were selected from the Sexually Transmitted Diseases Centre of Peking Union Medical College Hospital. Venous blood was collected and serum was extracted. T. paUidum antibodies in whole blood, anticoagulant whole blood, and serum were detected using 4 recombinant T. pallidum antigen-based rapid tests. T. pallidum haemagglutination test （TPHA） was considered as the gold standard for the detection of T. pallidum specific antibodies in serum. The sensitivities and specificities of four methods were analyzed. Results The sensitivities and specificities of Abbott Determine Syphilis TP test, SD-BIOLINE Syphilis 3.0 test, VISITECT-SYPHILIS test, and Syphicheck-WB test for serum specimens were 100% and 98. 9%, 95.7% and 98.0%, 94.6% and 98.2%, 68.1% and 98.9% ; for whole blood were 74. 1% and 99. 5%, 87.9% and 99.4% , 73.2% and 99.7%, 64. 7% and 99.7%. The observed sensitivities of the 4 rapid diagnosis tests were not significantly different with TPHA （ P 〉 0.05 ）. Conclusions The 4 rapid tests show good performance and characteristics in the diagnosis of syphilis. Furthermore, they are more sensitive for serum specimens than whole blood.展开更多
To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. ...To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. pallidum by polymerase chain reaction (PCR) and subcloned into appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzyme digestion, the recombinant plasmid was transfected into HeLa cells using liposome, and the expressed protein was identified by immunocytochemistry and Western blotting. After verifying that the Tp92 antigen gene fragment could be expressed in HeLa cells, 100?μg of recombinant plasmids [pcDNA3.1(+)-Tp92], 100 μg of control plasmids [pcDNA3.1(+)] or 0.5 ml PBS buffer were administered in 3 groups of New Zealand white rabbits (6 rabbits/group), and the booster immunizations were employed at 2-week interval for 3 times. ELISA assay was used for the quantitative detection of the specific antibody in the sera of rabbits, and the proliferation response of spleen cells was detected by MTT assay. It was found that the target gene Tp92 segment about 2103 bp was obtained, and the DNA sequence of Tp92 gene constructed in pcDNA3.1 (+) vector was consistent with the published nucleotide sequence. The homologies of the nucleotide and putative amino acid sequences of Tp92 gene between T.pallidum subsp. pallidum Nichols and various pathogenic treponeme strains were 95.5%-100%. The analysis of immunocytochemistry and Western blotting showed that Tp92 gene segment constructed in pcDNA3.1(+) vector could express a fusion protein with a calculated molecular mass of 77 kDa in HeLa cells and the expressed protein could react with positive blood serum from syphilis patient. The specific antibody IgG titers were observed and the highest titer was 1∶1024 in rabbits after 3 times with pcDNA3.1(+)-Tp92. The proliferation response of spleen cells were significantly higher than that of rabbits injected with pcDNA3.1(+) ( P <0.05). The successful expression of the eukaryotic expression plasmid of Tp92 gene from T. pallidum was obtained in eukaryotic system and strong responses of humoral and cellular immunity was evoked by DNA vaccine of pcDNA3.1(+)-Tp92 in rabbits thus establishing a solid basis for the future studies in the biological activities and for the development of the syphilis DNA vaccine.展开更多
To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was ...To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was amplified from the complete genome of T.pallidum by PCR, subcloned into expression vector pQE32 to generate the recombinant plasmid pQE32/Tp0453, then expressed in E.coli M15 and analyzed by SDS/PAGE and Western blotting. The fusion protein expressed was purified with Ni-NTA affinity chromatography. Its immuno-reactivity was assayed by indirect ELISA, and the immunogenicity was determined by immunization with this fusion protein in New Zealand rabbits. In the present study, a fusion protein of molecular weight about 32 kDa was obtained. As demonstrated by Western blotting, the recombinant protein could react specifically with positive IgG sera of patients with syphilis, and the antibodies against T.pallidum in human sera were successfully detected by indirect ELISA. Both the sensitivity and specificity of ELISA based on the Tp0453 fusion protein as were 100% (30/30) when detected with control sera. In comparison with the results of IgG ELISA with those of TPPA. It was found that the sensitivity of ELISA was 96.8% and the specificity was 100%. The difference of ELISA and TPPA was not significant, and the concordance of results between ELISA and TPPA was 98.2%. In addition, specific humoral responses could be elicited by immunization with the recombinant fusion protein in New Zealand rabbits with a specific antibody titer of 1∶1280 after 3 successive doses of immunization. These results demonstrate that the expressed recombinant fusion protein shows excellent immuno-competence and provide foundation to develop a quick diagnostic kid applied to detect the presence of T.pallidum infections.展开更多
The Gpd gene was amplified from the genomic DNA of Treponema the appropriate site of pcDNA3.1 （ ＋ ） vector. The expression of pcDNA3. I was tested with Western blotting and technology of immunoeytochemisty. New mun...The Gpd gene was amplified from the genomic DNA of Treponema the appropriate site of pcDNA3.1 （ ＋ ） vector. The expression of pcDNA3. I was tested with Western blotting and technology of immunoeytochemisty. New munized with the eukaryotic expression recombinant pcDNA3, 1 （ ＋ ）-Gpd pallidum and cloned into （ ＋ ）-Gpd in Hel,a cells Zealand rabbits were imA fusion protein of C, pd with 4.1 kDa has been effectively expressed in HeLa cells, which were detected bv Western blotting and the immunocytochemistry techniques. The New Zealand rabbits were able to elicit the specific antibody after immunization with the nucleic acid vaccine. The antibody titer could reach as high as 1 ： 1024 after 2 weeks of the third injection; and the splenocytes proliferated evidently due to the Gpd protein stimulation. Both the antibody titer and the splenocytes proliferation were higher substantially than those of controls （ P 〈 0.01 ）. All above data will contribute to an experimental basis of further study of the biological function of Gpd protein as well as DNA vaccine for syphilis.展开更多
Objective: To evaluate the clinical application of multiplex PCR inthe detection of Treponema pallidum, Herpes simplex virus (HSV), andHaemophilus ducreyi. Method: Three standard strains were used to set up a multiple...Objective: To evaluate the clinical application of multiplex PCR inthe detection of Treponema pallidum, Herpes simplex virus (HSV), andHaemophilus ducreyi. Method: Three standard strains were used to set up a multiplexPCR (MPCR) for detecting syphilis, herpes genitalis, and chancroidsimultaneously. Samples from 122 patients with genital ulcer disease(GUD) were subjected to MPCR and the results were compared withthose of dark-field microscopy and TP serology, HSV antigen ELISA,and H. ducreyi culture. Results: In the 122 patients with GUD, MPCR identified 34 casesof T. pallidum infection, 40 cases of HSV infection, and 2 cases of mixedinfection of T. pollidum and herpes. No positive results of H. ducreyiwere found. The sensitivity of MPCR to T. pallidum and herpes was100% and 93.3%, respectively. The sensitivities of dark-field mi-croscopy and TP serology, HSV antigen ELISA, and H. ducreyi cul-ture was 35.3%, 50% and 100%, respectively. Conclusion: MPCR showed a relatively higher sensitivity for T.pallidum as compared with the routine techniques. Although its sensi-tivity for HSV was not as good as that of antigen ELISA, it also yieldeda high detection rate. MPCR can detect more than one pathogen. It issimple, quick, sensitive, and suitable for clinical use or epidemiologicalinvestigation.展开更多
Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Met...Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Methods: The Gpd gene was amplified from the genomic DNA of T.pallidum by polymerase chain reaction (PCR) and inserted into cloning vector pUCm-T. The inserted Gpd gene was subcloned into the appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzymes digestion, the recombinant plasmid was transfected into Hela cells using liposomes. The expressed protein was identified by immunocytochemistry and Western blot. Results: The target Gpd gene segment was approximately 1059bp. The DNA sequence of the Gpd gene contained in the pcDNA3.1(+) vector was consistent with the published nucleotide sequence. The homology of the nucleotide and putative amino acid sequences of the Gpd gene between T. pallidum subsp. pallidum Nichols and various pathogenic treponemal strains ranged from 98% to 100%. Immunocytochemistry and Western blot analysis showed that the constructed Gpd-pcDNA3.1(+) vector expressed a fusion protein with a calculated molecular mass of 41KDa in Hela cells and that the expressed protein reacted with the sera from syphilis patients. Conclusion: The successful construction and expression of the eukaryotic expression plasmid of the Gpd gene from T.pallidum provide a promising tool to further study the biological activity of T.pallidum and develop a DNA vaccine for syphilis.展开更多
Objective: To obtain recombinant Treponema pallidumsubsp pallidum (TP 17KD) lipoprotein in large quantities byamplification and to further purify antigens for laboratorydiagnosis of syphilis and development of a syphi...Objective: To obtain recombinant Treponema pallidumsubsp pallidum (TP 17KD) lipoprotein in large quantities byamplification and to further purify antigens for laboratorydiagnosis of syphilis and development of a syphilis vaccine. Method: The Tpp17 lipoprotein gene was amplified fromthe TP(strain Nichols), and then it was recombinated into aplasmid pMAL-2c and cloned within E coli l2-TB1. The hostbacteria containing recombinant plasmids were induced withIPTG. The Tpp 17KD lipoprotein gene was amplified by us-ing PCR and positive clones were screened with double diges-tion and PCR. Recombinant plasmids were transformed intoE. coli and the E coli carrying recombinant plasmids wereinduced. The expression of TP 17KD was detected by sodiumdedecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot. Results:Gel staining with Coomassie blue G-250 showedthat the induced E coli carrying recombinant plasmid couldproduce 60KD fusion protein at high levels. Gel scanningshowed that 17KD protein expression in E coli accounted for10% of total cellular protein. The recombinant protein antigenreacted with the sera of syphilis patients. Conclusion: Our study lays a cornerstone for developingnew techniques of laboratory diagnosis for syphilis and newvaccines. Preliminary clinical application showed that thefusion protein could be used for the diagnosis of syphilis.展开更多
Treponema is a Gram-negative anaerobic bacterium,among which the pathogenic Treponema can cause various diseases,such as venereal syphilis(Treponema pallidum),yaws(Treponema carateum),and oral diseases(Treponema denti...Treponema is a Gram-negative anaerobic bacterium,among which the pathogenic Treponema can cause various diseases,such as venereal syphilis(Treponema pallidum),yaws(Treponema carateum),and oral diseases(Treponema denticola and Treponema medium).Although different from conventional lipopolysaccharides,the extracellular glycoconjugate of Treponema may still be a potential antigen and provide a candidate for vaccine development.Hence,we completed the first chemical synthesis of Treponema medium ATCC 700293 tetrasaccharide precursor containing L-ornithine(L-Orn)and D-aspartic acid(D-Asp)derivatives.The efficiency of non-reducing end disaccharide formation has been improved by optimizing the assembly of the protecting groups in the donors and acceptors.Our[3+1]glycosylation strategy attempted to reduce the length of the acceptor to increase the nucleophilicity of the hydroxyl group,thereby improving the efficiency of synthesizing the target tetrasaccharide.The L-Orn derivative was introduced at the final stage due to its influence on the glycosylation stereospecificity and efficiency.Therefore,the successful introduction of two amino acid derivatives and the synthesis of a tetrasaccharide precursor with complex functional-group modifications have provided valuable insights for synthesizing other complex bacterial glycans.展开更多
Screening for maternal syphilis has been an essential component of routine antenatal screening tests in most countries for many years. This is not only because of the virulence of the spirochete which causes the infec...Screening for maternal syphilis has been an essential component of routine antenatal screening tests in most countries for many years. This is not only because of the virulence of the spirochete which causes the infection but also because of its vertical transmission rate and the potential severe adverse complications/morbidity that can result from its transmission to the fetus. Although the incidence of maternal syphilis and its fetal sequalae in low-income countries has been considerable for several years, the disease has been almost non-existent in high income countries with wide antenatal screening coverage and effective treatment programmes for Syphilis. The recent alarming increase in the incidence of maternal syphilis in high income countries has spawned a renewed public health interest in the infection, with several countries updating and strengthening public health guidance in an attempt to stem this dramatic trend. This is a short clinical update for the practising obstetrician on how to manage the antenatal patient with a positive syphilis screening test.展开更多
文摘Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.
基金Financially supported by Key grant from the Education Committee of Hunan Province (No. 02A046)
文摘Objectives： To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods： The immuno-dominant epitope of Tp0453 gene was amplified from the complete genome of T.pallidum by polymerase chain reactions （PCR）, subcloned into the expression vector Pqe32 to generate recombinant plasmid Pqe32/Tp0453, and was then expressed in E. coli M15. The fusion protein was purified with Ni-NTA affinity purification. Indirect ELISA was developed to detect human serum IgG antibody to T. pallidum. Results： The recombinant Tp0453 protein was successfully expressed and purified. The recombinant protein had a molecular weight of approximately 32KDa.Indirect ELISA to the recombinant protein was developed.Sixty control sera were tested by ELISA and yielded a sensitivity of 100% （30/30） and a specificity of 100% （30/30）. While testing for T. pallidum in human sera, the sensitivity and specificity of ELISA were 96.8% and 100%, respectively, when compared with TPPA test results. The concordance of results between the ELISA test and the TPPA test was 98.2%. Conclusion： The recombinant Tp0453 outer membrane protein elicited a strong immunoreaction to anti-T.pallidum IgG antibody and has great potential use in ELISA for the serodiagnosis of syphilis.
文摘Objectives: To develop a multi-nested polymerase chain reaction in an assay to detect early Treponema pallidum and Haemophilius ducreyi DNA in the swabs of genital ulcers. Methods: Four pairs of outer and inner primers, specific to the basic membrane protein gene of Treponema pallidum and to the 16s rRNA gene of H ducreyi were synthesized. The multi-nested PCR was developed and applied to detect Treponema pallidum and Haemophilus dicreyi in clinical swabs. Result: The two samples of standard strains of Haemophilus ducreyi and one Treponema pallidum were amplified and showed 309-bp rRNA gene of Haemophilus ducreyi and 506-bp DNA of Treponema palidum, respectively. Out of 51 samples of genital ulcer detected, 29 showed Treponema pallidum positive product and no Haemophilus ducreyi DNA was found. Conclusion: The multi-nested PCR for Treponema pallidum and Haemophilus ducreyi could be useful for early detection and distinguishing diagnosis between syphilis and chancroid.
基金Supported by the WHO project on rapid diagnosis of syphilis (RFA-SDI-2001-02)
文摘Objective To assess the sensitivity, specificity, and feasibility of 4 recombinant Treponema pallidum antigenbased rapid tests in the diagnosis of syphilis. Methods A total of 970 outpatients were selected from the Sexually Transmitted Diseases Centre of Peking Union Medical College Hospital. Venous blood was collected and serum was extracted. T. paUidum antibodies in whole blood, anticoagulant whole blood, and serum were detected using 4 recombinant T. pallidum antigen-based rapid tests. T. pallidum haemagglutination test （TPHA） was considered as the gold standard for the detection of T. pallidum specific antibodies in serum. The sensitivities and specificities of four methods were analyzed. Results The sensitivities and specificities of Abbott Determine Syphilis TP test, SD-BIOLINE Syphilis 3.0 test, VISITECT-SYPHILIS test, and Syphicheck-WB test for serum specimens were 100% and 98. 9%, 95.7% and 98.0%, 94.6% and 98.2%, 68.1% and 98.9% ; for whole blood were 74. 1% and 99. 5%, 87.9% and 99.4% , 73.2% and 99.7%, 64. 7% and 99.7%. The observed sensitivities of the 4 rapid diagnosis tests were not significantly different with TPHA （ P 〉 0.05 ）. Conclusions The 4 rapid tests show good performance and characteristics in the diagnosis of syphilis. Furthermore, they are more sensitive for serum specimens than whole blood.
基金This work was supported by Hunan Provincial Education Department (002A046) and Department of Public Health of Hunan province (B2003 085)
文摘To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. pallidum by polymerase chain reaction (PCR) and subcloned into appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzyme digestion, the recombinant plasmid was transfected into HeLa cells using liposome, and the expressed protein was identified by immunocytochemistry and Western blotting. After verifying that the Tp92 antigen gene fragment could be expressed in HeLa cells, 100?μg of recombinant plasmids [pcDNA3.1(+)-Tp92], 100 μg of control plasmids [pcDNA3.1(+)] or 0.5 ml PBS buffer were administered in 3 groups of New Zealand white rabbits (6 rabbits/group), and the booster immunizations were employed at 2-week interval for 3 times. ELISA assay was used for the quantitative detection of the specific antibody in the sera of rabbits, and the proliferation response of spleen cells was detected by MTT assay. It was found that the target gene Tp92 segment about 2103 bp was obtained, and the DNA sequence of Tp92 gene constructed in pcDNA3.1 (+) vector was consistent with the published nucleotide sequence. The homologies of the nucleotide and putative amino acid sequences of Tp92 gene between T.pallidum subsp. pallidum Nichols and various pathogenic treponeme strains were 95.5%-100%. The analysis of immunocytochemistry and Western blotting showed that Tp92 gene segment constructed in pcDNA3.1(+) vector could express a fusion protein with a calculated molecular mass of 77 kDa in HeLa cells and the expressed protein could react with positive blood serum from syphilis patient. The specific antibody IgG titers were observed and the highest titer was 1∶1024 in rabbits after 3 times with pcDNA3.1(+)-Tp92. The proliferation response of spleen cells were significantly higher than that of rabbits injected with pcDNA3.1(+) ( P <0.05). The successful expression of the eukaryotic expression plasmid of Tp92 gene from T. pallidum was obtained in eukaryotic system and strong responses of humoral and cellular immunity was evoked by DNA vaccine of pcDNA3.1(+)-Tp92 in rabbits thus establishing a solid basis for the future studies in the biological activities and for the development of the syphilis DNA vaccine.
文摘To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was amplified from the complete genome of T.pallidum by PCR, subcloned into expression vector pQE32 to generate the recombinant plasmid pQE32/Tp0453, then expressed in E.coli M15 and analyzed by SDS/PAGE and Western blotting. The fusion protein expressed was purified with Ni-NTA affinity chromatography. Its immuno-reactivity was assayed by indirect ELISA, and the immunogenicity was determined by immunization with this fusion protein in New Zealand rabbits. In the present study, a fusion protein of molecular weight about 32 kDa was obtained. As demonstrated by Western blotting, the recombinant protein could react specifically with positive IgG sera of patients with syphilis, and the antibodies against T.pallidum in human sera were successfully detected by indirect ELISA. Both the sensitivity and specificity of ELISA based on the Tp0453 fusion protein as were 100% (30/30) when detected with control sera. In comparison with the results of IgG ELISA with those of TPPA. It was found that the sensitivity of ELISA was 96.8% and the specificity was 100%. The difference of ELISA and TPPA was not significant, and the concordance of results between ELISA and TPPA was 98.2%. In addition, specific humoral responses could be elicited by immunization with the recombinant fusion protein in New Zealand rabbits with a specific antibody titer of 1∶1280 after 3 successive doses of immunization. These results demonstrate that the expressed recombinant fusion protein shows excellent immuno-competence and provide foundation to develop a quick diagnostic kid applied to detect the presence of T.pallidum infections.
文摘The Gpd gene was amplified from the genomic DNA of Treponema the appropriate site of pcDNA3.1 （ ＋ ） vector. The expression of pcDNA3. I was tested with Western blotting and technology of immunoeytochemisty. New munized with the eukaryotic expression recombinant pcDNA3, 1 （ ＋ ）-Gpd pallidum and cloned into （ ＋ ）-Gpd in Hel,a cells Zealand rabbits were imA fusion protein of C, pd with 4.1 kDa has been effectively expressed in HeLa cells, which were detected bv Western blotting and the immunocytochemistry techniques. The New Zealand rabbits were able to elicit the specific antibody after immunization with the nucleic acid vaccine. The antibody titer could reach as high as 1 ： 1024 after 2 weeks of the third injection; and the splenocytes proliferated evidently due to the Gpd protein stimulation. Both the antibody titer and the splenocytes proliferation were higher substantially than those of controls （ P 〈 0.01 ）. All above data will contribute to an experimental basis of further study of the biological function of Gpd protein as well as DNA vaccine for syphilis.
文摘Objective: To evaluate the clinical application of multiplex PCR inthe detection of Treponema pallidum, Herpes simplex virus (HSV), andHaemophilus ducreyi. Method: Three standard strains were used to set up a multiplexPCR (MPCR) for detecting syphilis, herpes genitalis, and chancroidsimultaneously. Samples from 122 patients with genital ulcer disease(GUD) were subjected to MPCR and the results were compared withthose of dark-field microscopy and TP serology, HSV antigen ELISA,and H. ducreyi culture. Results: In the 122 patients with GUD, MPCR identified 34 casesof T. pallidum infection, 40 cases of HSV infection, and 2 cases of mixedinfection of T. pollidum and herpes. No positive results of H. ducreyiwere found. The sensitivity of MPCR to T. pallidum and herpes was100% and 93.3%, respectively. The sensitivities of dark-field mi-croscopy and TP serology, HSV antigen ELISA, and H. ducreyi cul-ture was 35.3%, 50% and 100%, respectively. Conclusion: MPCR showed a relatively higher sensitivity for T.pallidum as compared with the routine techniques. Although its sensi-tivity for HSV was not as good as that of antigen ELISA, it also yieldeda high detection rate. MPCR can detect more than one pathogen. It issimple, quick, sensitive, and suitable for clinical use or epidemiologicalinvestigation.
基金Focal point financial assistance entry(002A046)Department of Education, Hunan Province Scientific Research Foundation(B2003-085) Department of Public Health, Hunan province.
文摘Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Methods: The Gpd gene was amplified from the genomic DNA of T.pallidum by polymerase chain reaction (PCR) and inserted into cloning vector pUCm-T. The inserted Gpd gene was subcloned into the appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzymes digestion, the recombinant plasmid was transfected into Hela cells using liposomes. The expressed protein was identified by immunocytochemistry and Western blot. Results: The target Gpd gene segment was approximately 1059bp. The DNA sequence of the Gpd gene contained in the pcDNA3.1(+) vector was consistent with the published nucleotide sequence. The homology of the nucleotide and putative amino acid sequences of the Gpd gene between T. pallidum subsp. pallidum Nichols and various pathogenic treponemal strains ranged from 98% to 100%. Immunocytochemistry and Western blot analysis showed that the constructed Gpd-pcDNA3.1(+) vector expressed a fusion protein with a calculated molecular mass of 41KDa in Hela cells and that the expressed protein reacted with the sera from syphilis patients. Conclusion: The successful construction and expression of the eukaryotic expression plasmid of the Gpd gene from T.pallidum provide a promising tool to further study the biological activity of T.pallidum and develop a DNA vaccine for syphilis.
文摘Objective: To obtain recombinant Treponema pallidumsubsp pallidum (TP 17KD) lipoprotein in large quantities byamplification and to further purify antigens for laboratorydiagnosis of syphilis and development of a syphilis vaccine. Method: The Tpp17 lipoprotein gene was amplified fromthe TP(strain Nichols), and then it was recombinated into aplasmid pMAL-2c and cloned within E coli l2-TB1. The hostbacteria containing recombinant plasmids were induced withIPTG. The Tpp 17KD lipoprotein gene was amplified by us-ing PCR and positive clones were screened with double diges-tion and PCR. Recombinant plasmids were transformed intoE. coli and the E coli carrying recombinant plasmids wereinduced. The expression of TP 17KD was detected by sodiumdedecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot. Results:Gel staining with Coomassie blue G-250 showedthat the induced E coli carrying recombinant plasmid couldproduce 60KD fusion protein at high levels. Gel scanningshowed that 17KD protein expression in E coli accounted for10% of total cellular protein. The recombinant protein antigenreacted with the sera of syphilis patients. Conclusion: Our study lays a cornerstone for developingnew techniques of laboratory diagnosis for syphilis and newvaccines. Preliminary clinical application showed that thefusion protein could be used for the diagnosis of syphilis.
基金the National Natural Science Foundation of China(22325803,22077052,22277042,22107037,22177041,22207042)the China Postdoctoral Science Foundation(2021M691279)the National Key R&D Program of China(2023YFC2308000).
文摘Treponema is a Gram-negative anaerobic bacterium,among which the pathogenic Treponema can cause various diseases,such as venereal syphilis(Treponema pallidum),yaws(Treponema carateum),and oral diseases(Treponema denticola and Treponema medium).Although different from conventional lipopolysaccharides,the extracellular glycoconjugate of Treponema may still be a potential antigen and provide a candidate for vaccine development.Hence,we completed the first chemical synthesis of Treponema medium ATCC 700293 tetrasaccharide precursor containing L-ornithine(L-Orn)and D-aspartic acid(D-Asp)derivatives.The efficiency of non-reducing end disaccharide formation has been improved by optimizing the assembly of the protecting groups in the donors and acceptors.Our[3+1]glycosylation strategy attempted to reduce the length of the acceptor to increase the nucleophilicity of the hydroxyl group,thereby improving the efficiency of synthesizing the target tetrasaccharide.The L-Orn derivative was introduced at the final stage due to its influence on the glycosylation stereospecificity and efficiency.Therefore,the successful introduction of two amino acid derivatives and the synthesis of a tetrasaccharide precursor with complex functional-group modifications have provided valuable insights for synthesizing other complex bacterial glycans.
文摘Screening for maternal syphilis has been an essential component of routine antenatal screening tests in most countries for many years. This is not only because of the virulence of the spirochete which causes the infection but also because of its vertical transmission rate and the potential severe adverse complications/morbidity that can result from its transmission to the fetus. Although the incidence of maternal syphilis and its fetal sequalae in low-income countries has been considerable for several years, the disease has been almost non-existent in high income countries with wide antenatal screening coverage and effective treatment programmes for Syphilis. The recent alarming increase in the incidence of maternal syphilis in high income countries has spawned a renewed public health interest in the infection, with several countries updating and strengthening public health guidance in an attempt to stem this dramatic trend. This is a short clinical update for the practising obstetrician on how to manage the antenatal patient with a positive syphilis screening test.
