Objective To study the relation of type Ⅱ topoisomerase gene mutation and mechanism of Ureaplasma urealyticum resistant to quinolones. Methods 13 isolates of Uu resistant to 6 quinolones were selected from 184 clinical isolates by using broth dilution method, and their gyrA, gyrB, parC, parE were amplified by PCR.After sequencing, results were compared with the nucleotide sequence of susceptible reference strain. ResultsMICs of resistant Uu isolates were 4 to 32 fold higher than susceptible reference strain counter parts; sequence comparison revealed a C to A change at 87nt of gyrA QRDR led to the substitution of aspartic acid by glutamic acid and a C to T change at 50nt of parC QRDR led to the substitution of serine by leucine, with no amino acid change observed in GyrB and ParE. Conclusion These results suggest that a C to A change at 87nt of gyrA QRDR and a C to T change at 50nt of parC QRDR are associated with quinolone resistance of Uu.

Objective To investigate the distribution of subtypes of T. pallidum (TP) in Hengyang and Jiangmen regions. Methods Eighty-five specime ns taken from patients with suspected chancre collected in Hengyang and Jiangmen from February 2002 to January 2004 were screened by a PCR targeted TP polA gene , and then the arp gene and tpr gene were amplified from TP positive specimens. The PCR products of tpr gene were digested by restriction endonuclease MSe I. Th e sizes of the arp gene and the restriction fragment length polymorphism (RFLP) of the tpr gene were analyzed for subtyping. Results Of 69 TP-positive specime ns, 57 could be subtyped, and 10 subtypes were found. Among them, 26 (45.6%) wer e subtype 14d, and other subtypes included 10d(1), 12a(3), 12g(2), 13d(6), 14a(5 ), 14b(2), 14f(6), 15d(5) and 16d(1). No significant difference of the distribut ion of TP subtypes between Hengyang and Jiangmen was found. Conclusion Multipl e T.pallidum subtypes have been prevalent in Hengyang and Jiangmen, although the predominant subtype is 14d, there is no significant geographic heterogeneity be tween these two regions.

Objective To clone,express,and purify Tp 0319 outer membrane protein of Treponema pallidum and to develop an indirect ELISA for diagnosing syphilis.Methods The expression plasmid PQE32/Tp 0319 was conventionally constructed.The recombinant Tp 0319 protein was produced in E.coli M15 after induction by IPTG.The Tp 0319 protein was analyzed by SDS-PAGE and Western blotting,and then purified with Ni-NTA affinity chromatography.Indirect ELISA was developed to detect the syphilis antibody in human sera.Results The recombinant plasmid PQE32/Tp 0319 was constructed successfully and the fusion protein with relative molecular weight near 30 000 Dalton was revealed by SDS-PAGE.Western blotting proved that the recombinant protein specifically reacted with anti-Tp antibodies in sera from syphilis patients.The results of the indirect ELISA indicated the sensitivity and the specificity were both 100%.The concordance of 300 sera(150 from blood donors and 150 from syphilis patients)detected in parallel by the ELISA and the TPPA was 95.3%.Conclusions The data suggest that the prepared recombinant protein Tp 0319 of Treponema pallidum has high immunoreactivity.The recombinant protein can be used to develop ELISA kit for diagnosing syphilis.

Objective To investigate whether nuclear transcription factor κB(NF-κB) through Toll-like receptors 2(TLR2) was activated by lipid-associated membrane proteins(LAMPs) of Mycoplasma genitalium.Methods LAMPs were extractded and THP-1 cells were stimulated.The activation of NF-κBp65 was detected by ELISA and the expression of TLR2 mRNA was detected by RT-PCR.Effects of TLR2 neutralizing antibody on LAMPs induced the activation of NF-κBp65 was analyzed by ELISA.After LAMPs stimulated 293T cells with the co-transfection pFLAG-TLR2,pNF-κB-luc,pRL-TK,the activity of NF-κB firefly luciferase and pRL-TK Renilla luciferase were detected by the dual-luciferase reporter gene,to analyzed the role of TLR2-mediated NF-κB activation by LAMPs in 293T cells.Results The activation of NF-κBp65 was mediated in LAMPs induced THP-1 cells and was significantly increased by LAMPs in a dose dependent manner.when LAMPs was 4.0 μg/ml,the activation of NF-κBp65 was the highest level.TLR2 mRNA expression was up-regulated by LAMPs in THP-1 cells.TLR2 neutralizing antibody could inhibit the activation of NF-κB by 60% in LAMPs stimulated THP-1.NF-κB fluorescence was significantly increased by co-transfection pFLAG-TLR2 in a dose-dependent manner. ConclusionMycoplasma genitalium-derived lipid-associated membrane proteins activate NF-κB via TLR2 and the activation of TLR2-mediated play an important role in pathogenic process of LAMPs.

To localize and characterize the hypothetical protein CT358 in the chlamydial infected cells. CT358 gene from the Chlamydia trachomatis (C. trachomatis) serovar D genome was amplified and cloned into the pGEX and pDSRedCI vectors. The recombinant plasmid pGEX-CT358 was constructed and expressed as GST fusion proteins. The GST-CT358 fusion protein was used to immunize mice to raise the antibodies, which specifically recognized CT358 without eross-reacting with other unrelated proteins. The antibodies were then used to localize the endogenous CT358 protein and determine the expression pattern in Chlamydial infected cells using an indirect immunofluorescence assay (IFA). Meanwhile, pDSRedC 1-CT358 recombinant plasmid was transfected to HeLa cells to evaluate the effect of CT358 expression on the subsequent chlamydial infection. The hypothetical protein CT358 was identified in the inclusion membrane of C. trachomatis-infected cells for the first time,and it was detected as early as 12 h after C. trachomatis infection and remained in the inclusion membrane throughout the rest of the infection cycle. Cytosolic expression of CT358 via a transgene failed to affect the subsequent ehlamydial infection. These observations together have demonstrated that CT358 is a newly identified chlamydial inclusion membrane protein, giving the potentially importance for further understanding the mechanisms of chlamydial intracellular parasitism.

OBJECTIVE To study the expression situation of esp gene and the relativity between esp gene and resistance of Enterococcus faecalis derived-infection.METHODS The E.faecalis esp gene was amplified by PCR,then it was cloned and sequenced;the minimum inhibitory concentration(MIC) of five antimicrobicals against E.faecalis was determined by agar dilution method.RESULTS The positive rate of esp gene in 61 strains of E.faecalis was 42.6%;the positive rate of esp gene in the resistant strains against ampicillin,ciprofloxacin,high-level gentamicin,and erythromycin was 33.3%,54.8%,70.6%,and 51.0%,respectively,and the positive rate of esp gene in the susceptible strains against above 4 antibacterials was 43.6%,30.0%,7.4%,and 8.3%,respectively.CONCLUSIONS Incidence of esp gene does not correlate with the resistance of E.faecalis against ampicillin,but clearly correlate with the resistance of E.faecalis against ciprofloxacin,high-level gentamicin,and erythromycin;esp could become a marker of high-level gentamicin against E.faecalis derived-infection.

Objective To construct the recombinant plasmid containing the major outer membrane protein(MOMP) gene of Chlamydia trachomatis and expres s MOMP protein in E.coli BL21. Methods The MOMP gene was amplified by polymera se chain reaction from the genome of Chlamydia trachomatis serovar D. The amplif ied fragment was directly inserted into pUCm-T vector and verified by DNA sequen cing. MOMP gene was then subcloned into the prokaryotic expression vector pET-22 b(+). The recombinant protein of MOMP was purified by Ni-NTA affinity chromatogr aphy and identified by SDS-PAGE and Western blot. Results The MOMP gene, which is about 1 200 bp, was successfully amplified and cloned. The DNA sequence of t he cloned MOMP gene was the same as that published by the GenBank. SDS-PAGE anal ysis showed that the relative molecular weight of this fusion protein was about 47 kDa which was consistent with the theoretically predicted value, and the spec ificity of this recombinant protein was confirmed by Western blot. Conclusions The MOMP gene of Chlamydia trachomatis was successfully cloned and expressed in the prokaryotic expression system, which may lay the foundation for the developm ent of Chlamydia trachomatis vaccine.

Objective To express outer membrane protein 2(Omp2) of Chlamydia trachomatis, purify expressed products and study its immunity.Methods The target gene encoding Omp2 167—434 amino acid residues was amplified by PCR from C. trachomatis template DNA. The targeted DNA fragment was cloned into expression vector pET28b(+) and introduced into competent E. coli BL21(DE3) cell. Recombinant Omp2aa_ 167 ～aa_ 434 was expressed after induction by IPTG and analyzed by SDS-PAGE and Western blot, purified with Ni-NTA-His affinity chromatography. The rOmp2aa_ 167 ～aa_ 434 was used to immune rabbits for immunogenicity assessment.Results Restriction enzymes cleavage analysis and DNA sequencing confirmed that the plasmid pET28b(+)/Omp2aa_ 167 ～aa_ 434 was correctly constructed. The 35.0?103 molecular weight pure protein, which specifically reacted with serum from C. trachomatis infected patient by Western blot, was obtained by optimizing the conditions for both expression and purification. The titer of serum antibodies was above 1∶1 280 as detected by ELISA.Conclusion The expressed product showed good immunity.

Objective To clone.and express Tp0453 outer membrane protein of Treponema pallidum and develop an indirect ELISA for sero diagnosis of syphilis. Methods The immuno-dominant epitope of Tp0453 was amplified by PCR and subcloned into the expression vector pQE32.The recombinant protein was expressed in E.coli M15 and purified with Ni-NTA affinity chromatography columns. Indirect ELISA was developed to detect the antibody to Tp in human sera.Results 60 control sera was tested by ELISA.The sensitivities was 100%(30/30), and the specificities was 100%. While detecting uninfected and infected T. pallidum human sera, the sensitivities of ELISA was 96.8% compared with the results of the TPPA tests, and the specificities was 100% when the results of ELISA was compared with those of the TPPA test. The concordance of results between the ELISA test and the TPPA test was 98.2%.Conclusion The recombinant Tp0453 outer membrane protein showed excellent immuno-reactive activity, and were suitable for development of ELISA for sero-diagnosis of syphilis.

To localize and characterize the hypothetical protein CT358 in the chlamydial infected cells.CT358 gene from the Chlamydia trachomatis(C.trachomatis) serovar D genome was amplified and cloned into the pGEX and pDSRedC1 vectors.The recombinant plasmid pGEX-CT358 was constructed and expressed as GST fusion proteins.The GST-CT358 fusion protein was used to immunize mice to raise the antibodies,which specifically recognized CT358 without cross-reacting with other unrelated proteins.The antibodies were then used to localize the endogenous CT358 protein and determine the expression pattern in Chlamydial infected cells using an indirect immunofluorescence assay(IFA).Meanwhile,pDSRedC1-CT358 recombinant plasmid was transfected to HeLa cells to evaluate the effect of CT358 expression on the subsequent chlamydial infection.The hypothetical protein CT358 was identified in the inclusion membrane of C.trachomatis-infected cells for the first time,and it was detected as early as 12 h after C.trachomatis infection and remained in the inclusion membrane throughout the rest of the infection cycle.Cytosolic expression of CT358 via a transgene failed to affect the subsequent chlamydial infection.These observations together have demonstrated that CT358 is a newly identified chlamydial inclusion membrane protein,giving the potentially importance for further understanding the mechanisms of chlamydial intracellular parasitism.