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Objective To investigate the prevalence of A2058G or A2059G mutation within 23S rRNA in Treponema pallidum (Tp) from primary syphilis patients chancre samples. Methods Simple PCR was used to screen the positive samples containing Tp DNA. Nested PCR was adopted to amplify the region of the Tp 23S rRNA and the purified amplicons were digested by restriction endonuclease MboⅡand Bsa I respectively and sequenced. Results 39 qualified samples were obtained from 43 chancre samples and all of them were found harboring the A2058G mutation, whereas the A2059G was not detected. Conclusion High frequency of the A2058G mutation within 23S rRNA implicated in macrolide resistance emerges in the circulating Tp in Hengyang. Therefore, macro-lide antibiotics such as azithromycin should be cautiously used as an optional therapy for syphilis.
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Objective To clone, express, purify and evaluate the immunoreactivity of the recombinant protein Tp0844 of Treponema pallidum (Tp), and to screen major Tp proteins with high host reactivity. Methods The Tp0844 gene sequence was obtained through bioinformatics analysis. A prokaryotic expression vector of the Tp0844 gene was constructed and transformed into E. coli BL21 followed by isopropyl-1-thio-β-D-galactopyranoside (IPTG)induction for the expression of the recombinant protein Tp0844. Nickel-NTA affinity chromatography columns were utilized to purify the recombinant protein, and Western blotting was performed to evaluate the reactivity of the recombinant protein with sera positive or negative for anti-Tp IgG antibodies. Results The recombinant prokaryotic expression vector PET-30a (+)-Tp0844 was successfully constructed. After IPTG induction, a soluble recombinant protein with a relative molecular mass of about 43 000 was highly expressed, and purified by affinity chromatography. Western blotting showed that the Tp0844 recombinant protein specifically reacted with anti-Tp IgG antibody-positive sera, but not with anti-Tp IgG antibody-negative sera. Conclusions The soluble recombinant protein Tp0844 has good immunoreactivity, and can serve as a candidate antigen for investigation into the pathogenesis of syphilis.
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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.
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Objective To investigate the immune response to and protective effect of a bivalent DNA vaccine expressing interleukin-2(IL-2)and Gpd proteins in New Zealand rabbits.Methods Seventy-two male New Zealand white rabbits were equally and randomly divided into 4 groups to be immunized with recombinant plasmids pcDNA3.1(+)/Gpd-IL-2(pcD/Gpd-IL-2),pcDNA3.1(+)/Gpd(pcD/Gpd),empty plasmid pcDNA3.1(+)(pcD)and phosphate buffered saline(PBS),respectively.Immunization was carried out by intramuscular injection at multiple sites with a 2-week interval for 3 times.On week 10 after the initial immunization,the rabbits were challenged intradermally with T.pallidum(Nichols strain).Enzyme-linked immunosorbent assay(ELISA)was used to quantify the serum level of anti-Gpd antibodies in the rabbits and the level of IL-2 and interferon(IFN-γ)in the supernatant of Gpd protein-stimulated spleen cells from the rabbits at different time pionts.MTT assay was conducted to detect the proliferation response of spleen cells collected from the rabbits on day 0,14,28,140 and 168 after the challenge.Results Compared with pcD and PBS,both the vaccines pcD/Gpd and pcD/Gpd-IL-2 elicited significantly higher levels of anti-Gpd IgG antibodies in rabbits at different time points during the vaccination and infection period,with the titers peaking at 1 ∶ 1024 and 1∶4096,respectively(both P ＜ 0.01).There were also significant differences in the serum levels of anti-Gpd IgG antibodies between the pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits at different time points(all P ＜0.01).The levels of IL-2 in the supematant of spleen cells from pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits on week 8 after the immunization were 110 ± 12.6 and 167 ± 15.7 μg/L respectively,and those of IFN-γwere 225 ± 17.6 and 447 ± 22.4 μg/L respectively,significantly higher than those in that from the other two groups of rabbits(all P ＜ 0.01).Furthermore,an apparent proliferation response was observed in spleen cells from pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits with a higher stimulation index compared with pcD-and PBS-immunized rabbits(all P ＜ 0.01).Dark-field microscopic examination of early-stage infected lesions revealed that pcD/Gpd-IL-2-immunized rabbits had a lower detection rate(17.5％)of Tp from lesions,occurrence of ulcerative lesions(15％)and shorter curing time compared with pcD/Gpd-immunized rabbits.Conclusion The recombinant plasmid pcDNA3.1(+)/Gpd-IL-2 could induce protective humoral and cellular immune response more efficiently in rabbits.
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Objective To clone, express Tp0319 gene from Treponemapallidum (T. pallidum), and to assess the immunocompetence of recombinant protein. Methods The immuno-dominant region of Tp0319gene was chosen by computer analysis, amplified from T. pallidum complete genome by PCR, subcloned into the expression vector pQE32 to construct a recombinant plasmid, pQE32/Tp0319, which was then expressed in E. coli M15. The recombinant protein was purified with Ni-NTA affinity chromatography, and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blot. New Zealand rabbits were immunized with the recombinant protein, and the titer of anti-Tp0319 antibodies in sera from immunized rabbits were measured with indirect ELISA. Also, indirect ELISA with the recombinant Tp0319 as coating antigen was performed to detect the anti-Tp0319 antibody in sera from 200 normal human controls and 200 patients with syphilis. Results The prokaryotic expression vector pQE32/Tp0319 was constructed successfully, and the recombinant protein Tp0319 with a molecular weight of about 30 000 was attained. Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the specific antibody titer was more than 1: 10 240 after immunization for 3 times. Western blot proved that the recombinant protein could specifically react with anti-T. pallidum IgG antibody-positive sera. Indirect ELISA was successfully developed with the recombinant Tp0319, and detected antibodies to T. pallidum in control sera with a sensitivity and specificity of 100% (40/40), respectively. Compared with T. pallidum particle agglutination (TPPA) assay, the sensitivity and specificity of the indirect ELISA were 92.6% and 100%, respectively, in the detection of T. pallidum in sera from patients and controls, and the concordance between the indirect ELISA and TPPA was 96%. Conclusions The prepared recombinant protein shows a satisfactory immunocompetence, which may lay a foundation for its further application in the serodiagnosis of syphilis.
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Objective To construct a recombinant plasmid encoding Tp0821,a membrane lipoprotein of T. pallidum,express and purify this protein,and to evaluate its immunocompetence.Methods The recombinant plasmid pQE32/Tp0821 was constructed and induced to express the corresponding protein.Then,New Zealand rabbits were immunized with purified recombinant protein to prepare polycional antibodies,and the titer of polyclonal antibody was determinated.Indirect ELISA was developed with the recombinant protein of T. pallidum as coating antigen to detect 80 control sera and 150 FTA-ABS-positive sera.Results The recombinant plasmid pQE32/Tp0821 was constructed and a fusion protein with expected molecular weight was expressed.Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the antibody titer reached 1:6400.Compared with FTA-ABS test,the indirect ELISA showed a sensitivity and specificity of 92.6%and 98.6%,respectively,in the detection of control and clinical sera.Conclusion The recombinant protein Tp0821 shows excellent immunocompetence,which can be applied to the serological diagnosis of syphilis.
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Objective To express Tp0751 laminin-binding adhesion of Treponema pallidum (T. pallidum) ,and assess the immunocompetence. Methods The Tp0751 ORF without upstream non-cod-ing region was ligated into the expression vector pET-28a( + ), and expressed in E. coli R2566. Its immuno-gen was analyzed by Western blot and ELISA. Results A fusion protein with molecular weight about 26×103 was attained after expression and purification. Western blot proved that the recombinant protein can specifically react with T. palliclum IgG positive sera. Specific humoral response were elicited after introducing recombinant protein in Zealand rabbit and the specific antibody titer was above 1:10 2400 detected by indi-rect ELISA. Conclusion The expressed recombinant protein showed excellent immunoeompetence, and the results lay the foundation for the research on its function to T. paUidum infection.
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OBJECTIVE To investigate the antimicrobial activity in vitro of cefoperazine/sulbactam for non-fermentative Gram-negative bacilli and analyze its antimicrobial capacity in vitro in comparison with imipenem and other antimicrobial drugs.METHODS The 362 clinical strains of non-fermentative Gram-negative bacilli were collected from Jun 1,2008 to Jun 1,2009.The drug resistance of the strains to 9 antibiotics was detected.RESULTS The primary non-fermentative Gram-negative bacilli were Pseudomonas aeruginosa,Acinetobacter baumannii,Stenotrophomonas maltophilia and Burkholderia cepacia in our hosipal.The resistant rates to cefoperazine/sulbactam were 29.2%,20.8%,25.0% and 27.3%,respectively.Cefoperazine/sulbactam was better than imipenem and meropenem and far better than piperacillin/tazobactam.CONCLUSIONS The resistant rate of non-fermentative Gram-negative bacilli is very high,and it shows multi-drug resistance.The results show cefoperazine/sulbactams is useful for non-fermentative Gram-negative bacilli infection with multi-drug resistance.
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OBJECTIVE To evaluate the spectrum of imipenem-resistant Pseudomnas aeruginosa and the production of metallo-?-lactamase.METHODS The clinical strains of P.aeruginosa were collected from Jan to Dec 2007.The results of antimicrobial susceptibility tests and detection of metallo-?-lactamase were analyzed.Antimicrobial susceptibility tests were performed by K-B methods;the production of metallo-?-lactamase was tested by CAZ-EDTA synergy method.RESULTS Sixty strains were isolated,imipenem-sensitive and resistant strains were 40(66.7%) and 20(33.3%),respectively,and 7 strains with metallo-?-lactamase were detected.Among imipenem-resistant strains,at least 90.0% strains were resistant to meropenem,gentamicin,tobramycin,ciprofloxacin and SMZ-TMP;at least 80.0% strains were resistant to piperacillin and piperacillin/tazobactam;50.0% strains were resistant to ceftazidime and cefepime;polymyxin E was less resistant than others.Twenty strains were resistant to at least 3 antimicrobial agents,which was obviously higher than 27.5% of imipenem-resistant strains.CONCLUSIONS The resistance rate of imipenem-resistant P.aeruginosa is higher than imipenem-sensitive ones.The production of metallo-?-lactamase is one of the mechanisms of P.aeruginosa resistance to imipenem and shou1d be detected carefully,which could help us medicate reasonably in clinic and avoid using medicine which could induce and strengthen the resistance.
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OBJECTIVE To investigate the distribution of clinical bacterial isolates and the change in antibiotic resistance spectrum in our hospital from 2005 to 2007.METHODS Data of bacterial susceptibility testing of clinical isolates from the Second Affiliated Hospital in of University of South China from 2005 to 2007 were collected and analyzed by software WHONET25.Results were assessed according to the National Committee for Clinical Laboratory Standards(NCCLS) of America issued in 2005.RESULTS The amount of Gram-negative bacteria decreased and of Gram-positive bacteria increased during this period.The proportion of coagulase negative Staphylococcus(CNS) had been increasing and reached 21.7% in 2007.The proportions of Staphylococcus aureus decreased from 17.6% in 2005 to 13.0% in 2007.Escherichia were the top two bacteria in 2007.The drug resistance rate of staphylococci against penicillin and erythromycin was more than 92.2% and 52.2%,respectively.The oxacillin resistance rate of CNS was 74.5%,significantly higher than that of S.aureus(16.5%).Drug resistance rate of Enterococcus to vancomycin was 1.1%.Gram-negative bacteria were found resistant to meropenem and imipenem.The resistance rate to ampicillin of Klebsiella and Escherichia was very high.CONCLUSIONS The variation of drug resistance and distribution of clinical bacterial isolates in our hospital are related to the improper use of antibiotics.It is very important to select antibiotics correctly according to the results of antibiotics susceptibility tests.
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By using methods of forecast research,system science,comparison research and abstract research,main deficiencies and practical problems in requisitioning civilian medical equipment are analyzed.It is suggested that we should regard the health service's demand as direction,the preparation as premise,the information construction as fundamentality,the lawmaking as guarantee,to improve and perfect the work about the requisition of civilian medical equipment and improve the quality and benefit of mobilization.
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The dependability of medical equipment directly affects the quality and benefit of health service. The medical equipment dependability of our army apparently lags behind foreign armies. It is urgent for us to improve the dependability of the medical equipment.Our army must innovate the research and production as well as purchase of the medical equipment; enhance the inherent dependability and the use dependability of the medical equipment, actualize research on non-effectiveness of the medical equipment, improve the production design and enhance the skill of the operators.
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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.
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OBJECTIVE To develop the strong and the specific multi-epitope antigen for the exploiting diagnosis of Treponema pallidum.METHODS The immuno-dominant epitopes of Tp0453 and Tp17 were amplified by PCR respectively,and subcloned into the expression vector pQE32 to generate multi-epitopes recombinant plasmid pQE32/Tp0453-17.The recombinant protein was expressed in Escherichia coli M15.The immunoresponse of recombinant fusion protein was analyzed by Western blot.RESULTS The multi-epitopes recombinant plasmid was successfully constructed,enzyme digestion analysis and sequencing showed that the inserted target genes were Tp0453 and Tp17 gene,compared with the gene reported by GenBank,it had 100% similarity;SDS-PAGE analysis showed the recombinant plasmid could be expressed in M15,its relatively molecular mass(Mr) of expressed product was about 52.0?103.The Western blot result showed the recombinant protein could be recognized by anti-T.pallidum positive serum.CONCLUSIONS The expressed multi-epitopes recombinant antigen showed excellent immunoresponse.The results lay the foundation for research on development of quick diagnostic kit applying to detection of T.pallidum infection.
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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.