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 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.

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.

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.

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.

In recent years,the course of pathogenic biology of Nanhua University has made great achievements because the discipline characteristics has been stressed.Therefore,the reputation of Nanhua University is improved with the elevation of this discipline.The development patterns of Nanhua University provide some experience for the building and development of general university.

Colleges are responsibile for training high-quality professionals and top talents.College teachers are decisive for teaching quality.As a result,to strengthen the faculty construction and establish excellent college teaching team are important for the improvement of the quality of personnel training.

With the development of basic disciplines such as molecular biology,immunology,cell biology and so on. the pathogen biology research do not stop at the organ and cellular level,but go deep into the protein and gene level. It is a great boost to the deep studies of pathogen biology in diagnosis,treatment,pathogenesis,prevention and epidemiology.

Objective To localize and characterize the plasmid protein pORF5 in the Chlamydia trachomatis(Ct) infected cells. Methods The open reading frame encoding for pORF5 protein from the Ct plasmid was amplified and cloned into the pGEX-6p vector. The recombinant plasmid pGEX-pORF5 was transformed into XL1-blue E. coli to express fusion protein with the glutathione-s-transferase (GST). After purified with Glutathione Sepharose 4B beads, the pORF5 fusion protein was used to immunize mice to make monoclonal and polyclonal antibody. The antibodies were used to localize the endogenous pORF5 protein and detect the expression pattern in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). At the same time, ELISA was used to determine whether pORF5 plasmid protein was expressed and immunogenic during Ct infection in humans. Results pORF5 was detected a dominant signal in the cytosol of the Chlamydia-infected cells with a pattern similar to that of anti-CPAF. pORF5 also appeared in the RBs and EBs in small quantity. Athough pattern was similarly, pORF5 did not overlap with CPAF. pORF5 protein was strongly recognized antiserum in an ELISA. Conclusion The pORF5 plasmid protein was identified as a secreted protein with good immunogenicity, pORF5 gene was to express the endogenous target protein during human infection.