ObjectiveTo study the effect of a recombinant plasmid encoding mouse interleukin 2 (mlL-2) on the immunogenicity of DNA vaccine against Chlamydia trachomatis(Ct) serovar E.Methods BALB/c mice were divided into 4 groups to be intramuscularly inoculated with blank plasmid(negative control group),DNA vaccine against Ct serovar E(DNA vaccine group),DNA vaccine against Ct serovar E and a recombinant plasmid containing mIL-2(combination group),and inactivated Ct serovar E elementary bodies (positive control group),respectively.The immunological effects were evaluated by posterior foot pad thickness,proliferation level of spleen lymphocytes,serum level of IL-4 and interferon (IFN)-γ in mice,and the capability to clear Ct genital tract infection.ResultsThe proliferation index of spleen lymphocytes in the combination group and positive control group was similar(3.64 ± 0.41 vs.3.77 ± 0.34),but was significantly different from that in the blank control group and DNA vaccine group (1.37 ± 0.21 and 2.52 ± 0.30).The serum level of IL-4 was(38.49 ± 12.24) pg/ml in the positive control group,significantly higher than in the negative control group,DNA vaccine group and combination group ((25.37 ± 18.93),(24.75 ± 8.49),(21.74 ± 6.43) pg/ml,respectively).With respect to the serum level of IFN-γ,the combination group and positive control group were similar ((1923.3 ± 518.1) pg/ml vs.(2712.5 ± 887.2) pg/ml),but were significantly different from the negative control group and vaccine group((310.8 ± 160.7) pg/ml and(601.3 ± 357.9) pg/ml).Six days after Ct challenge,the exfoliated cells from genital tract were positive for Ct culture in the negative control group,but negative in the other 3 groups.ConclusionIL-2 genetic adjuvant can enhance the immune response,especially Th1 type response,induced by the DNA vaccine against Ct serovar E.

Objecfive To detect serum antibodies to Pmp in patients with urogenital Chlamydia trachomatis infection and to assess the relationship between Pmp and urogenital C.traehomatis infection.Methods Twenty healthy adults and 77 patients with urogenital C. trachomatis infection were recruited into this study.A 3-month foilow-up was carried out in 43 patients,who were classified into persistent infection group(n=19)and negative-conversion group(n=24).Western-blot was performed to detect serum antibodies to Pmp in all subjects.Results The positivity rate of anti-Pmp antibodies was 90.20% (71/77) in patients,significantly higher than that in the normal controls[20% (4/20),P<0.05].All the 9 types of anti-Pmp antibodies were detected in patients with a varying positivity rates,which were 61.04% (47/77),88.31% (68/77),63.63% (49/77),28.57% (22,77),63.63% (49/77),75.32% (58/77),62.34% (48/77),77.92% (60/77)and 70.13% (54/77) for antibodies against PmpA,PmpB,PmpC,PmpD,PmpE,PmpF,PmpG,PmpH and PmpI respectivelyThe prevalence was highest for anti-Pmp B antibodies and lowest for anti-Pmp D antibodies.There was no significant difference in the positivity rate of anti-Pmp antibodies between persistent infection group and negativeconversion group.Conclusions Anti-Pmp antibodies could be generated in patients infected with C. trachomatis.The immunogenicity of different Pmps is different,and the immunoprotective activity of Pmps is rather weak.Individual differences exist in serum anti-Pmp antibodies among patients.Nine types of Pmps are expressed in patients with urogenital C. trachomatis infection.

Objective To get phagc's capsid Vp3 gene and protein of guinea pig inclusion conjunctivitis (GPIC) chlamydia. Methods The genome DNA was extracted from the φCPG1 phage.The full sequence of Vp3 gene was amplified by polymerase chain reaction (PCR) from the above genome DNA. The Vp3 gene was digested by restriction endonuclease and then inserted into prokaryotic plasmid vector pET30a (+). The recombinant plasmid was transformed into E. coil BL21, and was identified by restriction endonuclease, PCR and sequencing. The E. coil BL21 with expected recombinant plasmid was induced and the expressed recombinant Vp3 protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, then purified by agarose gel. Results The recombinant gene was sequenced and proved to be 447 bp which was consistent with the φCPG1 Vp3 gene sequence in GenBank. A 25 000 capsid protein was expressed and confirmed by SDS-PAGE and Western blot. The purified protein was obtained. Conclusion The capsid Vp3 protein of φCPG1 is successfully expressed and purified, which is helpful for the further study on its mechanism and clinical applications.

Objective To investigate the relationship of point mutations in ERG11,TAC1,MRR1 and UPC2 genes to fluconazole resistance in Candida albicans.Methods This study included 11 fluconazole-resistant Candida albicans strains.DNA was extracted from these Candida isolates by urea pyrolysis method.PCR was carried out to amplify the full-length EGR11 gene,as well as the corresponding fragments of TAC1,MRR1 and UPC2 genes followed by DNA sequencing.Results Eight mutations were detected in the ERG11 gene,including D116E,Y132H,Y132F,K143Q,T229A,E266D,G448E and G464S.Among them,K143Q was a novel mutation and had not been reported.A G648D mutation was detected in the UpC2 gene.No mutations were detected in the TAC1 or MRR1 gene.Conclusions There is a definite association between fluconazole resistance and ERG11 mutations,while further research is warranted to clarify the relationship between the mutations of TAC1,MRR1,UPC2 genes and resistance to fluconazole.

Objective To clone, express and purify Chlamydia trachomatis polymorphic membrane protein (Pmp G), and to identify its immunogenicity. Methods The Pmp G gene of C. trachomatis serotype E was amplified by PCR, cloned into prokaryotic expression vector PET30a (+). The positive recombinant was transformed into the bacterium E coli (BL-21), identified by enzyme digestion, PCR amplification and gene sequencing. Then, it was induced to express followed by the identification of expression product with SDS-PAGE and Western blotting. The purified protein was used to immunize BALB/C mice to test its immunogenicity. Results PCR produced a 1092 bp-sized DNA fragment, which had a sequence consistent with that of PmpG gene of C. trachomatis E type in the GenBank database. The molecular weight of expression product was 55 kD, which was proved to be the expected size, and Western Blotting confirmed it to be the specific protein. Moreover, special antibodies to PmpG were induced to be generated by mice immunized with the purified protein. Conclusions The constructed prokaryotic expression vector for PmpG is expressed successfully in E. coli, and the expression product shows immunogenicity.

Objective To investigate the influences of lysozyme on the mRNA expressions of MMP-1,-12 and lysyl oxidase(LOX)in cultured fibroblasts in vitro.Methods Primarily cultured fibroblasts isolated from human skin were treated with three concentrations(0.1×10~(-8),1×10~(-7)mol/L)of lysozyme followed by another 24-hour cuhure.Subsequently,total RNA was extracted from the fibroblasts and subjected to RT-PCR for the detection of MMP-1,-12 and LOX mRNA.Results There was a significant difference in the mRNA expressions of MMP-1,-12 and LOX among the fibroblasts treated with the three concentrations of lysozyme (F=6.98,4.44,5.24,respectively,all P<0.05).SNK-q test showed that untreated fibroblasts differed signifi-cantly from those treated with lysozyme of 1×10~(-7) mol/L in the mRNA expression of MMP-1 and MMP-12 (P<0.05),and from those treated with iysozyme of 1×10~(-7) mol/L.and 1×10~(-8)mol/L in the mRNA expres-sion of LOX(both P<0.05),whereas no significant difference was ohserved between fibroblasts treated with lysozyme of 1×10~(-8) mol/L and untreated fibrohlasts or those with lysozyme of 1 x 10~(-7)mol/L in the mRNA expression of MMP-1 and MMP-12.or between fibroblasts treated with lysozyme of 1 x 10~(-8)mol/L and those with that of 1×10~(-7) mol/L in the expression of LOX (all P>0.05).Conclusions Lysozyme upregulates the mRNA expression of MMP-1 and MMP-12 but downregulates the mRNA expression of LOX in cultured fibro-blasts in vitro.

Objective To investigate specific immune responses in mice induced by recombinant major outer membrane protein(rMOMP)of C.trachomatis serovaf E.Methods Thirty-six female BALB/cmice aged 3 to 4 weeks Were divided into three groups.i.e.,adjuvant group vaccinated、with purified rMOMP and Freund's adjutant,solitary group vaccinated with rMOMP only and control group vaccinated with phosphate buffered saline(PBS).All the mice were intramuscularly vaccinated on week 0,2 and 4.Blood samples and vaginal washes were obtained from these mice on week 6,then,mice were challenged with elementary body(EB)of C.trachomatis serovar E at the footpad followed by the observation of delayed hypersensitivity.On week 7.mice were genitally infected with C.trachomatis EB;one week later,blood samples and vaginal washes were obtained again;six weeks later,spleen lymphocytes were isolated from the mice and stimulated bv C.trachomatis or ConA followed by the detection of cell proliferation with MTT assay.In vitro neutralization assay was also performed.ELISA was used to determine the titers of Chlamydia-specific IgO antibody in sera and IgA antibody in vaginal washes,as well as the level of IFN-γ in culture supernatant of lymphocytes and sefa of mice.Vaginal swabs were collected after genital challenge and subjected to C.trachomatis culture.Results The absorbance at 405 ms of Chlamydia-specific IgG antibody and proliferation index of lymphocytes were 0.641±0.059 and 5.085±1.291.respectively,in mice immunized with rMOMP and Frennd's adjuvant.significantly higher than those in mice immunized with rMOMP only(0.424±0.015 and 3.123 ±0.840.both P<0.05).The thickness of right hind footpad increased by 0.324±0.054 mm and 0.272±0.064 mm,respectively,in solitary group and adjuvant group,respectively,with significant difference between the two groups(P<0.05).A significant increase was also observed in the adjuvant group compared with the control group in the above three parameters(all P<0.01).Conclusion The rMOMP of C.trachomatis could efficiently induce Chlamydia-specific humoml and cellular immune responses in mice.

Objective To evaluate the susceptibility of Chlamydia trachomatis clinical isolates to rifampin, and assess the relationship between rpoB mutations and antibiotic resistance in them.Methods A microculture method was used to determine the minimal inhibitory concentration (MIC) of rifampin in 52 Chlamydia trachomatis clinical isolates.The rpoB gene was amplified from all the clinical isolates and a standard strain of Chlamydia trachomatis followed by single-strand conformation polymorphism (SSCP)analysis.Sequencing of PCR products was carried out for two clinical isolates.Results No rifampin-resistant strain was found among these clinical isolates.The MIC of rifampin varied from 0.004 to 0.030 mg/L Neither SSCP analysis nor sequencing showed rpoB mutations.Conclusions No rpoB mutations were found in Chlamydia trachomatis isolates from patients unresponsive to rifampin.The unresponsiveness to rifampin may be attributed to multiple factors.

Objective To test cross immune responses induced in rhesus monkeys immunized with the recombinant major outer membrane protein(rMOMP).Methods Six rhesus monkeys were divided into three groups:the group vaccinated with purified rMOMP and Freund's adjutants,the group vaccinated with Freund's adjutants only and the control group vaccinated with PBS.All of the rhesus monkeys vaccinated intramuscularly at 0,2,4 weeks.Two weeks after the last time,The IFN-γand Chlamydia-specific antibody titers in sera,which were determined by ELISA,lymphocyte proliferation assay were performed by MTT,and observ the delayed hypersensitivity and in vitro neutralization assays.Results The result of the monkeys immunized with rMOMP and Freund's adjuvant:the specific immune responses can be observed.The in vitro neutralization and lymphocyte proliferation assays were observed better in the same group.Conclusion After being vaccinated with rMOMP,the monkeys can develop strong and effective Chlamydia-specific cross immune responses.

Objective To assess polymerase chain reaction(PCR)combined with restriction fragment length polymorphism(RFLP)and gene sequencing technologies in the detection and typing of HPV DNA.Methods Tissue specimens were collected from skin diseases and venereal disease in perianal or genitals.PCR was performed with HPV DNA general primers(MY09/11)in tissue samples. Positive fragments of HPV DNA were purified and digested by restriction enzymes.The digested fragments were typed by po]yacrylamide gel electrophoresis(PAGE).The Resultswere verified by direct sequencing.Results In 50 clinical samples there were 35 HPV DNA positive,including 26 from patients with condyloma acuminatum,8 from patients with bowenoid papulosis,and 1 from patients with squamous cell carcinoma.In HPV DNA positive samples,19 were HPV6,3 were HPV11,8 were HPV16,4 were HPV6 and HPV 11,and I was HPV62.Sequencing Resultswere in accordance with the PCR-RFLP Results .Conclusion PCRRFLP method is effective in the detection and typing of HPV DNA.