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 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 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 construct DNA vaccine containing MOMP gene of Chlamydia trachomatis and to observe immune response in mice. Methods Mice of 4 - 6 weeks old were immunized with pcDNA3.1-MOMP or pcDNA3.1 intramuscularly at a dose of 100 μg. Booster immunizations were employed at 2-week interval for two times. Specific antibody in the sera of mice and the level of IFN-γ in murine spleen lymphocyte supernatant were detected by ELISA. The proliferation response of spleen cells was detected by MTT assay. Results Significant specific antibody titers were observed and the highest titer was 1: 1 024 in mice after three times immunization with pcDNA3.1-MOMP. The proli-feration response of spleen cells were significantly higher than that of mice injected with pc DNA3.1. IFN-γ reached(532.0 + 45.4)pg/mL in immunized mice. Conclusion Strong responses of humoral and cellular immunity can be evoked by DNA vaccine of pcDNA3.1-MOMP in mice.

Objective To evaluate the value of a Treponema pallidum (TP) recombinant protein TP0993 in the serodiagnosis of syphilis.Methods A bioinformatics method was used to obtain the sequence of TP0993 gene.The open reading frame (ORF) without upstream non-coding region of TP0993 gene was ligated into the expression vector PET-28a (+),which was then transformed into Escherichia coli Rosetta.Isopropyl-β-d-thiogalactoside (IPTG) was used to induce the expression of TP0993 protein.The expressed protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography.Western blot was performed to evaluate the immunoantigenicity of the protein.New Zealand rabbits were immunized with the recombinant protein for immunogenicity evaluation.Indirect enzyme linked immunosorbent assay (ELISA) was developed by using the purified recombinant protein to coat microwell plates.Anti-TP antibodies were detected by the established ELISA and TP particle agglutination assay (TPPA) in 480 clinical serum samples.Results The prokaryotic expression vector PET-28a (+)-0993 was successfully built,and a fusion protein with a relative molecular weight of about 34 000 Da was attained after IPTG-induced expression and purification.Western blot proved that the recombinant protein could specifically react with clinical sera positive for anti-TP IgG antibodies.Specific humoral response was elicited in New Zealand rabbits by the recombinant protein.Compared with TPPA,the established indirect ELISA showed a sensitivity of 88.3％ and a specificity of 85.8％.There was a consistency of 86.5％ between the indirect ELISA and TPPA.Conclusion The expressed recombinant protein showed favorable immunocompetence,and may serve as a candidate antigen for serodiagnosis of syphilis.

Quality is the lifeline of graduate education and the tutor team plays an important role in postgraduate training. To control the selection of tutors strictly, to implement the combination of openness and stability in tutor team, and to carry out academic exchanges actively are the keys to construct an excellent tutors staff and to ensure the quality of graduate student training.

Objective To clone and express the immunodominant domain of the chlamydial proteaselike activity factor(CPAF) from Chlamydophila psittaci(Cps) and evaluated the diagnosing value of the recombinant protein in Cps infection.Methods The immunodominant region epitope of CPAF (CPAFm,A196-A450)from Cps was chosen according to bioinformatics analysis and references.The specific primer was designed and the gene was amplified by PCR and then ligated into a pGEX6p-2 vector.Recombinant protein was induced to express by IPTG and analyzed by SDS-PAGE and Western blot.Indirect EL1SA method of serological diagnosis was established with the reorganization protein as coating antigen.One hundred and eighty sera samples from ducks with respiratory tract infection symptoms were detected with the established indirect ELISA and a commercial ELISA-kit to assess the value of the recombinant protein in serodiagnosis.The results were further identified with Western blot.Results Prokaryotic expression vector pGEX6p-2/CPAFm was constructed and a 54x103 fusion protein was attained.The indirect ELISA method was established with reorganization protein for envelope antigen.Using the indirect ELISA to detect Cps lgG positive and negative reference sera,the sensitivity and specificity were both 100％ (20/20).And the recombinant protein has no cross reaction with either Chlamydophila pneumoniae or Chlamydophila trachomatis.The concordance rate between the indirect ELISA and Western blot to 180 ducks sera samples was 100％,while the concordance rate of the commercial ELISA kit was 77.5％-95.0％.Conclusion The prepared recombinant protein of the CPAF immunodominant region epitope gene from Gps can highly benefit on developing new indirect ELISA as methods to detect specific anti-Cps antibodies.

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.

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.