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Objective To screen and identify the in vivo-induced antigen Tp0462 of Treponema pallidum (Tp),and to evaluate its value for clinical serological diagnosis of syphilis.Methods Genomewide DNA was extracted from the Tp Nichols strain,and polymerase chain reaction (PCR) was performed to amplify the Tp0462 gene.A recombinant plasmid pET30a (+)-Tp0462 was constructed and transfected into the Escherichia coli (E.coli) Rosetta (DE3) strain.Recombinant protein Tp0462 was abundantly expressed,purified and identified.A total of 18 New Zealand rabbits were randomly and equally divided into 3 groups:viable Tp-incubating group incubated with viable Tp in the testes,inactived Tp-incubating group incubated with ultraviolet irradiation-killed Tp in the testes,and control group receiving no treatment.After incubation,blood samples were collected at different time points,and the sera were isolated for identification of characteristics of in vivo-induced antigen Tp0462.Enzyme-linked immunosorbent assay for Tp0462 (Tp0462-ELISA),Treponema pallidum particle agglutination assay (TPPA),preliminary syphilis screening ELISA and rapid plasma reagin (RPR) card test were applied in 336 clinical serum samples from patients with syphilis,so as to preliminarily evaluate the value of Tp0462 for the diagnosis of syphilis.Results The optimum conditions for expression of the recombinant plasmid Tp0462-pET30a(+) in E.coli Rosetta (DE3) strains were the treatment with 0.5 mmol/L isopropyl thiogalactoside (IPTG) on a shaker at 180 rpm for 4 hours.In the viable Tp-incubating group,the serum level of specific anti-Tp0462 antibody sharply increased from week 2,and went steady after week 5.However,the specific anti-Tp0462 antibody maintained a low level in the inactived Tp-incubating group and the control group.The viable Tp-incubating group showed a significantly higher level of specific anti-Tp0462 antibody compared with the inactived Tp-incubating group and the control group (both P ＜ 0.05),while no significant difference was observed between the inactived Tp-incubating group and the control group (P =0.256).The level of anti-Tp92 antibody was significantly higher in the viable Tp-incubating group and the inactived Tp-incubating group than in the control group (P ＜ 0.05),while there was no significant difference between the viable Tp-incubating group and the inactived Tp-incubating group (P =0.127).Compared with TPPA,the sensitivity,specificity,consistency rate and area under the curve (AUC) of Tp0462-ELISA for the diagnosis of syphilis were 91.7％,98.8％,95.2％ and 0.997 respectively.Tp0462-ELISA was consistent to preliminary syphilis screening ELISA and RPR with a Kappa coefficient of 0.846 and 0.293,respectively.Conclusion Tp0462-ELISA has shown evidently higher sensitivity and specificity in the serodiagnosis of syphilis,and Tp0462 can serve as promising antigens for the diagnosis of syphilis.
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Objective To evaluate immune protective effects of Treponema pallidum(Tp)pcD/Tp92 DNA vaccine delivered through different inoculation routes against Tp-induced skin infection in New Zealand rabbits. Methods A total of 108 New Zealand rabbits were randomly and equally divided into 6 groups:A1 and A2 groups treated with intramuscular injection of empty plasmids pcD and pcD/Tp92 DNA vaccine respectively for 2 sessions, B1, B2, C1 and C2 groups firstly treated with intramuscular injection of the pcD/Tp92 DNA vaccine for 1 session for primary immunization, then receiving nasogastric feeding with pcD/Tp92 DNA vaccine, pcD/Tp92 DNA vaccine+cytosine-phosphate-guanine(CpG)oligodeoxynucleotide (ODN), and recombinant Tp92 protein, and recombinant Tp92 protein+CpG ODN respectively for booster immunization. Enzyme-linked immunosorbent assay(ELISA)was conducted to detect the serum level of anti-Tp92 IgG antibody at week 0, 2, 4, 6, 8 after immunization, the SIgA level in the nasopharyngeal region and vaginal mucosa at week 8 after immunization, as well as levels of interleukin-2(IL-2)and interferon-γ (IFN-γ)in the culture supernatant of rabbit spleen cells at week 8 after immunization, and methyl thiazolyl tetrazolium(MTT)assay was performed to estimate proliferative activity of rabbit splenic lymphocytes in three rabbits from each group. At week 10 after immunization, other 15 rabbits from each group were subcutaneously inoculated with Tp standard strain, and changes of skin lesions at the inoculation site during early-stage infection were observed and recorded. Results At week 8 after immunization, the C2 group showed significantly higher serum level of anti-Tp92 IgG antibody(1.825 ± 0.175), supernatant levels of IL-2 (154.7 ± 14.6)and IFN-γ(277.4 ± 24.4), and proliferative activity of T cells(3.57 ± 0.24)compared with the A2(1.372 ± 0.322, 112.3 ± 13.4, 232.8 ± 25.3, 3.08 ± 0.22, respectively, all P<0.05), B1(0.893 ± 0.297, 76.6 ± 21.5, 165.7 ± 22.6, 2.12 ± 0.14, respectively, all P<0.05)and B2(1.294 ± 0.124, 97.3 ± 18.7, 211.3 ± 24.6, 2.88 ± 0.18, respectively, all P<0.05)groups. In addition, effective immunoprotection was achieved in the C2 group with more production of mucosa-specific SIgA antibody, as well as the lowest Tp-positive rate (6.67%) and ulcer formation rate (6.67%) in skin lesions at the inoculation sites. Conclusion The effective vaccination strategy, namely intramuscular injection of the pcD/Tp92 DNA vaccine for primary immunization followed by nasogastric feeding with mucosal adjuvant CpG ODN combined with recombinant Tp92 protein for booster immunization, can induce the strongest mucosal immune responses and immune protective effects.
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Objective To investigate the clinical distribution and antimicrobial resistant characteristics of Pseudomonas aeruginosa(PA)in the northern area of Guangdong.Provide the reference for clinical to prevent infection and reasonable choice of antibiotics and reduce the production of drug resistance strains.Methods The separation and identification of PA were performed by conventional methods during the data of drug 2013 and 2014.The data of sensitivity test of PA were analyzed by WHONET 5.6 and SPSS19.0 softwares.Results The 584 strains PA were mainly distributed in ICU,department of orthopaedics and respiratory medicine.Specimens were mainly from sputum and wound secretion.The detection of PA to 12 antibacterial agents showed different resistance.The antimicrobial with highest resistance was the gentamicin and lower resistance rates to fluoroquinolones,carbapenems,enzyme inhibitors.And a downward trend was shown in drug resistance to CIP,FEP,LEV,SCF.Conclusion PA mainly cause lung and wound infection,especially those old patients that come from ICU,department of orthopaedics and respiratory medicine.Although the drug resistance rates of PA to the commonly used antibiotics are relatively low,The clinicians should reasonably use antibiotics so as to reduce the resistant strains,especially the produce of MDR-PA and PDR-PA.
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Objective To investigate the resistance of clinical isolates from Yuebei People’s Hospital so as to provide the ref-erence for clinical use of antibiotics.Methods To comprehensively analyze the drug resistance,AST was performed with K-B or MIC method to 2 527 strains of clinical isolates and the data were analyzed by WHONET 5.4 and SPSS19.0 softwares according to the breakpoints of CLSI 2012 standard.Results MRSA accounted for 30.9% in Staphylococcus aureus iso-lates,MRCNS accounted for 76.2% incoagulase negative Staphylococcus isolates ,no detection of vancomycin or linezolid re-sistant strains.The antimicrobial drug resistance of Enterococcus faecium was significantly higher than that of Enterococcus faecalis ,no detection of glycopeptide and linezolid resistant strains in Enterococcus faecalis ,but one linezolid resistant strains and two teicoplanin resistant strains in Enterococcus faecalis .Enterobacteriaceae remain highly sensitive to carbapen-em antibiotics,ESBLs produced strains in Escherichia coli ,Klebsiella pneumoniae were 43.9% and 37.5% respectively,five polymyxin B resistant strains were detected in Pseudomonas aeruginosa ,and 63.7% of MDRO rate in Acinetobacter bau-mannii .Conclusion Drug resistance of clinical pathogenic bacteria remain seriously in hospital,clinical application of antibi-otics should be reasonable based on the result of drug sensitivity.
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To predict and identify the dominant B‐cell epitopes of conserved region of Treponema pallidum repeat protein F (TprFN ) and provide the basis for development of polyvalent epitope‐based syphilis vaccine ,the amino acid sequence of TprFN was obtained from GenBank and analyzed with comprehensive meta‐analysis Mobyle ,ABCpred and IEDB online software .The peptides containing predicted epitopes were artificially synthesized . To obtain and measure the titers of antibodies against TprFN ,New Zealand rabbits were immunized with recombinant protein TprFN expressed in E .coli and identified by Western blot (WB) .Sera from TprFN‐immunized rabbits ,syphilis patients ,and normal human and normal rabbits were used to deter‐mine the immunoreactivity and specificity of 7 predicted peptides of TpFN by indirect ELISA .Comprehensive meta‐analysis of online software showed that P1 (43‐62AA) ,P2(57‐71AA) ,P3(81‐88AA) ,P4(89‐103AA) ,P5(125‐138AA) ,P6(231‐251AA) and P7(268‐279AA) might be the B‐cell epitopes .A protein was expressed in a soluble form and identified as TpFN by WB .The ELISA indicated that P1 and P3 were active with TprFN‐immunized rabbit sera and syphilis patient sera but not with negative control sera .These results indicate that P1 and P3 are the potential dominant B‐cell epitopes .
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Breaking through the traditional teaching model of medical microbiology,establishing the teaching philosophy of the subject to professional features and the core of combination with form as a breakthrough,promoting the intrest in active learning and the improvement of comprehensive ability of nursing profession students.
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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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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.
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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 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.