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Objective:To investigate the effect of recombinant lipoproteins of Brucella outer membrane protein 16, 19 (L16 and L19) on the expression of immune regulatory factors in human monocytic leukemia cell line (THP-1 cells). Methods:THP-1 cells activated with phorbol ester (PMA) were used as an in vitro experimental cell model, and a group design was used to co-culture L16, L19 and THP-1 cells (L16 stimulated group, L19 stimulated group), respectively. THP-1 cells activated with PMA were used as the control group. When co-cultured for 4 hours, immunofluorescence staining (IFS) and Western blotting were used to detect whether L16 and L19 entered the cells, respectively; when co-cultured for 12, 24 hours, real-time fluorescent quantitative PCR was used to measure the mRNA expression levels of interferon regulatory factor 1 (IRF-1) and trans activator protein of major histocompatibility complex class Ⅱ (CⅡTA); Western blotting was used to detect the protein expression levels of T cell immunoglobulin mucin-3 (Tim-3) and γ interferon receptor 1 (IFNGR1). Results:When co-cultured for 4 hours, L16 and L19 were observed entering THP-1 cells in the L16 stimulated group and L19 stimulated group, respectively. When co-cultured for 12 hours, the expression level of IRF-1 mRNA in the L16 stimulated group (0.16 ± 0.15) was significantly lower than that in the control group (1.00 ± 0.00, P < 0.05). When co-cultured for 24 hours, the expression level of CⅡTA mRNA in the L16 stimulated group (0.17 ± 0.10) was significantly lower than that in the control group (1.00 ± 0.00, P < 0.05). When co-cultured for 12 and 24 hours, there were no statistically significant differences in the expression levels of IRF-1 and CⅡTA mRNA between the L19 stimulated group and the control group ( P > 0.05). Western blotting results showed that there were statistically significant differences in the expression levels of INFGR1 and Tim-3 protein among the control group, L16 stimulated group, and L19 stimulated group after co-cultured for 12 and 24 hours ( F = 50.92, 6.80, 148.73, 156.57, P < 0.05). Among them, when co-cultured for 12 hours, the expression level of INFGR1 protein in the L16 and L19 stimulated groups were significantly lower than that in the control group, and the L19 stimulated group was higher than the L16 stimulated group ( P < 0.05), and the expression level of Tim-3 protein in the L19 stimulation group was higher than that in the control group ( P < 0.05). When co-cultured for 24 hours, the expression level of INFGR1 protein in the L16 and L19 stimulated groups were lower than that in the control group, and the L19 stimulated group was higher than that in the L16 stimulated group ( P < 0.05); and the expression level of Tim-3 protein in the L16 stimulated group was higher than that in the control group and L19 stimulated group ( P < 0.05). Conclusions:Brucella L16 can downregulate the expression levels of IRF-1 and CⅡTA mRNA in THP-1 cells. Both L16 and L19 can downregulate IFNGR1 and upregulate Tim-3 protein expression levels.
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@#Objective To develop a colloidal gold immunochromatographic test strip for rapid and accurate detection of Pseudomonas aeruginosa(P.aeruginosa,Pa).Methods After bioinformatics analysis of Pa outer membrane protein OprF,the gene sequence with abundant antigenic determinants and high intraspecific homology was chemically synthesized,and then connected to pET-28a(+)vector to construct the expression vector pET-28a-OprF,which was transformed into E.coli BL21(DE3)and induced by IPTG. The recombinant OprF protein was purified by Ni Sepharose~(TM)6 Fast Flow and used to immunize two female BALB/c mice for 3～4 times by multi-point subcutaneous injection in the back at the first immunization and intraperitoneal injection at subsequent immunizations. The monoclonal antibodies were screened by animal cell fusion technique,and the colloidal gold immunochromatographic test strip for rapid detection of Pa was prepared by using monoclonal antibody and double antibody sandwich immunochromatography technique. The specificity,sensitivity and stability of the test strip were evaluated.Results Two monoclonal antibodies,Pa-1# and Pa-2#,were obtained with the titer of 1∶409 600,and both of them recognized OprF specifically. The prepared colloidal gold immunochromatographic test strip showed a sensitivity of 1. 0×10~6CFU/mL and had no cross reaction with 9 common respiratory pathogens with a good stability.Conclusion The prepared colloidal gold immunochromatographic test strip can detect Pa rapidly within 15 min,with high specificity and good stability.
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Objective:High-throughput screening to obtain small molecular compounds against Gram-negative bacilli by targeting BamA outer membrane protein.Methods:The sybyl-X2.1 software was used to perform high-throughput virtual screening of small molecular compounds in Chemdiv compound library based on the molecular docking. The top 150 hits by high-throughput screening were re-screened through in vitro biological experiments. The top 4 small molecules with obvious antibacterial activity were selected for in-depth molecular docking analysis, and the small molecule 8308-0401 with the highest docking score was selected for further experiments. The antibacterial effect of 8308-0401 combined with rifampicin was tested by checkerboard assay. Finally, the affinity between 8308-0401 and BamA was tested by plasma surface resonance assay. Results:The docking score of the top 150 hits calculated by high-throughput virtual screening had a mean value of 5.63. In vitro biological experiments showed that small molecules 8308-0401, 8365-1335, C066-2507 and L582-0346 exhibited strong antibacterial activity. Among those molecules, 8308-0401 showed the highest molecular docking score, and synergistic antibacterial activity against both types of strains and clinical isolates when combined with rifampicin. 8308-0401 has a strong affinity to BamA with binding a constant of 182 μmol/L. Conclusion:The small molecule 8308-0401 exerts antibacterial activity against Gram negative bacilli by targeting the outer membrane protein BamA.
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@#Abstract：Objective To investigate the feasibility of outer membrane protein C（OmpC）as a protein presenting platform targeting antigen to the surface of outer membrane vesicle（OMV）. Methods The recombinant expression plasmid containing ompC gene fragment and Staphylococcus aureus EsxA antigen gene（esxA gene）was constructed，transformed to competent E. coli BL21（DE3），inducedbyIPTG，andanalyzedforexpressedproductby 12%SDS⁃PAGE. Thetotalproteinofrecombinant strain OMV was analyzed by 12% SDS⁃PAGE，and the localization of fusion protein on the surface of OMV was detected by Western blot and Flow NanoAnalyzer. Results The recombinant expression plasmid containing ompC gene and esxA gene was constructed correctly as proved by sequencing. 12% SDS⁃PAGE showed that the fusion protein OmpC⁃EsxA had a relative molecular mass of about 57 000，which was consistent with the expected size，while the total protein of OMV showed multiple target protein bands，indicating that recombinant strain OMV was successfully extracted. The fusion protein OmpC⁃ EsxA on the surface of recombinant strain OMV specifically bound to mouse antibody against His⁃Tag，and OMVs labeled with fluorescent antibody were detected by Flow NanoAnalyzer. Conclusion OmpC may be used as a protein presenting plat⁃ form to locate antigen to OMV surface，which was expected to be applied in the development of antigen presentation vaccine. Keywords：Outer membrane protein C（OmpC）；Protein presentation；Outer membrane vesicle（OMV）
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We optimized the expression and purification of outer membrane proteins SpaO and LamB from Salmonella typhi. We investigated various factors in the expression and purification processes, including the use of isopropyl ß-d-1 thiogalactopyranoside (IPTG), imidazole, and urea. First, PCR amplification was carried out on SpaO and LamB genes. The genes were then cloned in pTZ57R/T, and then expressed in pET28a vector and transformed into Escherichia coli BL21 (DE3). Gene insertion was confirmed by enzymatic digestion with NdeI and XhoI. Inclusion bodies expressing recombinant SpaO and LamB were induced with 200 and 400 µL 0.5 mM IPTG, respectively. The formed protein inclusion bodies were then isolated from the pellet and solubilized in IB buffer containing 8 M urea for SpaO and 6 M urea for LamB. Proteins were refolded by dialysis in 3M urea. Purified proteins with nickel-nitrilotriacetic acid affinity chromatography and eluted with buffer containing 250 mM imidazole for SpaO and 150 mM imidazole for LamB. Theâ¯protein expression profiles were analyzed by SDS-PAGE, which identified the 33 and 49 kDa bands corresponding to rSpaO and rLamB. Western blotting Purification was carried out by nickel affinity resin with 250 mM and 150 mM imidazole for rSpaO and rLamB and refolded through stepwise dialysis with anti-His tag antibodies confirmed their expression. These optimized methods can be used to generate recombinant proteins for the development of future vaccines.
Subject(s)
Salmonella typhi , Membrane ProteinsABSTRACT
ABSTRACT@#In recent years, antimicrobial resistance (AMR) has become a global public health concern. The growth of resistant bacteria is increasing dramatically, while the number of new antibiotics accessible is decreasing. This is especially true in the case of Pseudomonas aeruginosa, an important causative agent of healthcare-associated infections. The ability of P. aeruginosa to survive in different environments and on medical devices has made it more resistant to antibiotics. This causes bacteremia in hospitalized patients, ventilator-associated pneumonia, catheter-associated urinary tract infections and wound infections, particularly in patients with severe burns, bed ulcers and immunocompromised individuals. The rise in the AMR rate in both developed and developing countries may be attributed to a number of factors such as variations in the standard health care, large population, awareness about antibiotic resistance, inadequate training on rationale antibiotic usage and inadequate infection control facilities in many hospitals. The emergence of Extensive Drug Resistance (XDR) and Pan Drug Resistance (PDR) among organisms that cause various infections leads to increased treatment costs, morbidity and mortality, leaving no therapeutic options. This review highlights the different mechanisms of antibiotic resistance, including intrinsic and acquired resistance, which are frequently observed in P. aeruginosa.
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Pseudomonas aeruginosa , Drug Resistance, MicrobialABSTRACT
Objective:This study is designed to investigate the toxicity of lipoprotein (L16) and non-lipoprotein (U16) of Brucella outer membrane protein (OMP) 16 on osteoblasts. Methods:Recombinant L16 and U16 proteins were prepared by using prokaryotic expression system of Escherichia coli ( E. coli) BL21 (DE3) and purified by Ni column. Using group design, mouse osteoblasts (MC3T3 cells) were co-incubated with L16 and U16, respectively. Brucella lipopolysaccharide (LPS) stimulus was used as the positive control, and cells without any stimulation were used as the negative control. Incubation time was 24 h. The activity of co-incubated MC3T3 cells were detected by CCK-8; the supernatant of cultured cells was collected and the release rate of lactate dehydrogenase (LDH) in the supernatant was detected by bioluminescence, and the virulence of L16 and U16 on MC3T3 cells was evaluated. Annexin Ⅴ-PE/7-AAD double staining flow cytometry was further used to analyze the apoptosis rate of MC3T3 cells, and the activation level of apoptosis executive protein Caspase-3 was detected by Western blotting (WB). Results:The activity of MC3T3 cells in L16 group [(56.16±1.63)%] was significantly lower than that in U16 and LPS groups [(97.02±1.44)%, (98.64±0.90)%, P < 0.01], the LDH release rate [(84.64±0.96)%] was significantly higher than that in U16 and LPS groups [(34.82±3.41)%, (26.75±1.95)%, P < 0.01]. Annexin Ⅴ-PE/7-AAD double staining results showed that the apoptosis rate was (46.45±2.19)% in L16 group, while the remaining groups were all less than 1%. WB results showed that activated Caspase-3 (cleaved-Caspase-3) existed in L16 stimulated cells, but not in U16 stimulated cells and LPS control cells. Conclusion:L16 can induce the apoptosis of osteoblasts and inhibit the proliferation of osteoblasts, but U16 has no obvious effect indicating that Brucella L16 with complete lipid structure is necessary for virulence effect.
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@#Treponema denticola is an important pathogenic Treponema pathogen in the human oral cavity. Early studies have found that Treponema denticola is closely related to the occurrence and development of periodontal diseases. With the development of technical methods in recent years, many studies have shown that Treponema denticola not only can participate in periodontal diseases through a variety of mechanisms but also can play an important role in the development of various oral diseases. Treponema denticola is detected in high concentrations in peri-apical diseases and peri-implant diseases, and its surface protein is also prevalent in oral tumor samples. This paper reviews the research progress of Treponema denticola in periodontal diseases, pulp peri-apical diseases, peri-implant diseases and oral tumors, and summarizes the relevant mechanisms. For example, Treponema denticola can cause immune regulation disorder, destroy the epithelial barrier, induce bone absorption, promote the occurrence and development of inflammation through a variety of surface proteins, including chymotrypsin-like protease complex (CTLP), major outer sheath protein (Mosp), Td92, and LOS. It can also escape complement-mediated killing effects through surface FhbB lipoproteins and promote the occurrence and development of oral tumors by regulating the tumor microenvironment. These theories provide a theoretical basis for further understanding the development of oral diseases, controlling the infection of Treponema denticola, and exploring more effective treatment strategies.
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Humans , Anti-Bacterial Agents , beta-Lactam Resistance , Computational Biology , Dysentery, Bacillary , Enterobacteriaceae , Epitopes , Epitopes, T-Lymphocyte , Membrane Proteins , Membranes , Shigella flexneri , Shigella , T-Lymphocytes , Vaccines , Vaccines, SubunitABSTRACT
Objective@#To establish a labeling method for bacterial outer membrane vesicle (OMV) based on luciferase reporting gene. @*Methods@#By utilizing the characteristics that high abundance of outer membrane protein A (OmpA) presented on the surface of bacterial OMV, the outer membrane protein-luciferase fusion protein was constructed to position the luciferase on the surface of the outer membrane vesicle, and the number of bacterial OMV was evaluated based on the luciferase activity. @*Results@#The OmpA-NanoLuc fusion protein expression vector was constructed successfully, and the outer membrane vesicles secreted by the subject strains after the expression of the fusion protein displayed stable luciferase activity. The number of bacterial outer membrane vesicles was semi-quantitative detrmined by measuring the activity of fluorescein enzyme. @*Conclusion@#A semi-quantitative method based on luciferase labeling was developed for the detection of extracellular vesicles, which could be used to evaluate the secretion level of specific strains.
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Campylobacter is a major cause of food-borne gastroenteritis worldwide. While mortality is low when people was infected with Campylobacter, morbidity imparted by post-infectious sequelae such as Guillain-Barré syndrome and irritable bowel syndrome is significantly noteworthy. Although fluoroquinolones and macrolides were the first line drug for the treatment of Campylobacter infections, there is a tough challenge in clinical treatment with high antimicrobial resistant rate and multi antimicrobial resistance arise. Based on the latest literature acquired in this work, we have chosen five classes of antibiotics always used in clinical, and discussed antibiotic resistance mechanisms and transmission of Campylobacter, in order to provide proper therapy both in the veterinary and human populations, and support basis data for the development of new drugs.
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Outer membrane proteins (OMPs) of Gram-negative bacteria constitute the first line of defense protecting cells against environmental stresses including chemical, biophysical, and biological attacks. Although the 43-kDa OMP (OMP43) is major porin protein among Bartonella henselae-derived OMPs, its function remains unreported. In this study, OMP43-deficient mutant B. henselae (Δomp43) was generated to investigate OMP43 function. Interestingly, Δomp43 exhibited weaker proliferative ability than that of wild-type (WT) B. henselae. To study the differences in proteomic expression between WT and Δomp43, two-dimensional gel electrophoresis-based proteomic analysis was performed. Based on Clusters of Orthologus Groups functional assignments, 12 proteins were associated with metabolism, 7 proteins associated with information storage and processing, and 3 proteins associated with cellular processing and signaling. By semi-quantitative reverse transcriptase polymerase chain reaction, increases in tldD, efp, ntrX, pdhA, purB, and ATPA mRNA expression and decreases in Rho and yfeA mRNA expression were confirmed in Δomp43. In conclusion, this is the first report showing that a loss of OMP43 expression in B. henselae leads to retarded proliferation. Furthermore, our proteomic data provide useful information for the further investigation of mechanisms related to the growth of B. henselae.
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Bartonella henselae , Bartonella , Gram-Negative Bacteria , Information Storage and Retrieval , Membrane Proteins , Membranes , Metabolism , Proteomics , Reverse Transcriptase Polymerase Chain Reaction , RNA, MessengerABSTRACT
Objective:To evaluate preliminarily immunocompetence and immunoprotection of a NMB0315 nucleic acid vaccine,a recombinant protein vaccine and a nucleic acid vaccine plus a recombinant protein vaccine against Neisseria meningitidis serogroup B in mice, and to provide reliable experimental basis for further exploration of the effective immunization methods and pathways of NMB0315 vaccine. Methods:The NMB0315 nucleic acid vaccine [ pcDNA3. 1 (+)/NMB0315 ] and recombinant protein vaccine(pET-30a/NMB0315)were prepared. Female BALB/c mice were inoculated with a NMB0315 DNA vaccine followed by boosting with recombinant protein NMB0315 through intramuscular and intraperitoneal immunization respectively. Next, humoral immunologic response and cellullar immunologic response were detected in female BALB/c mice by ELISA. The survival rate of BALB/c mice was used to evaluate immunoprotection of the vaccines in mice. Results:Specific IgG,IgG1,IgG2a,and sIgA,induced by the NMB0315 DNA vaccine(pNMB0315-CpG),protein NMB0315 vaccine(rNMB0315-FA),NMB0315 DNA vaccine prime-protein boost at week 8, were detected by indirect ELISA,the A450 values were up to(0. 505±0. 042,0. 513±0. 022,0. 342±0. 017,0. 250±0. 015),(0. 823± 0. 061,0. 807±0. 045,0. 596±0. 027,0. 450±0. 028)and(0. 694±0. 053,0. 711±0. 032,0. 455±0. 021,0. 386±0. 024)respectively, which was significantly higher than the PBS control(P<0. 05). The antibody level of protein vaccine was significantly higher than the nucleic acid vaccine group and combined immunization group ( P<0. 05 ) . The stimulation index and IFN-γ level of combined immunization group were significantly higher than the protein vaccine group and nucleic acid vaccine group(P<0. 05). The bactericidal titer of nucleic acid vaccine group, protein vaccine group and combined immunization group reached 1 :64, 1 :128 and 1 :128 respectively,and the protection rates were 70%,95% and 80% respectively. The IgG2a/IgG1 ratios of the nucleic acid vaccine group, the recombinant protein vaccine group and the combined immunization vaccine group were all less than 1 at week 2, 4, 6, 8. Conclusion:The humoral immunity effects( including mucosal immune) induced by the NMB0315 vaccines form high to low were as follows:the recombinant protein vaccine group, the combined immunization vaccine group, the nucleic acid vaccine;and the cellular immune effects from high to low were as follows:the combined immunization vaccine group, the nucleic acid vaccine group, the recombinant protein vaccine group;The protection effects induced by the NMB0315 vaccines in BALB/c mice within 72 hours from high to low were as follows:the recombinant protein vaccine group, the combined immunization vaccine group, the nucleic acid vaccine group.
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Objective:To evaluate preliminarily immunocompetence and immunoprotection of a NMB0315 nucleic acid vaccine,a recombinant protein vaccine and a nucleic acid vaccine plus a recombinant protein vaccine against Neisseria meningitidis serogroup B in mice, and to provide reliable experimental basis for further exploration of the effective immunization methods and pathways of NMB0315 vaccine. Methods:The NMB0315 nucleic acid vaccine [ pcDNA3. 1 (+)/NMB0315 ] and recombinant protein vaccine(pET-30a/NMB0315)were prepared. Female BALB/c mice were inoculated with a NMB0315 DNA vaccine followed by boosting with recombinant protein NMB0315 through intramuscular and intraperitoneal immunization respectively. Next, humoral immunologic response and cellullar immunologic response were detected in female BALB/c mice by ELISA. The survival rate of BALB/c mice was used to evaluate immunoprotection of the vaccines in mice. Results:Specific IgG,IgG1,IgG2a,and sIgA,induced by the NMB0315 DNA vaccine(pNMB0315-CpG),protein NMB0315 vaccine(rNMB0315-FA),NMB0315 DNA vaccine prime-protein boost at week 8, were detected by indirect ELISA,the A450 values were up to(0. 505±0. 042,0. 513±0. 022,0. 342±0. 017,0. 250±0. 015),(0. 823± 0. 061,0. 807±0. 045,0. 596±0. 027,0. 450±0. 028)and(0. 694±0. 053,0. 711±0. 032,0. 455±0. 021,0. 386±0. 024)respectively, which was significantly higher than the PBS control(P<0. 05). The antibody level of protein vaccine was significantly higher than the nucleic acid vaccine group and combined immunization group ( P<0. 05 ) . The stimulation index and IFN-γ level of combined immunization group were significantly higher than the protein vaccine group and nucleic acid vaccine group(P<0. 05). The bactericidal titer of nucleic acid vaccine group, protein vaccine group and combined immunization group reached 1 :64, 1 :128 and 1 :128 respectively,and the protection rates were 70%,95% and 80% respectively. The IgG2a/IgG1 ratios of the nucleic acid vaccine group, the recombinant protein vaccine group and the combined immunization vaccine group were all less than 1 at week 2, 4, 6, 8. Conclusion:The humoral immunity effects( including mucosal immune) induced by the NMB0315 vaccines form high to low were as follows:the recombinant protein vaccine group, the combined immunization vaccine group, the nucleic acid vaccine;and the cellular immune effects from high to low were as follows:the combined immunization vaccine group, the nucleic acid vaccine group, the recombinant protein vaccine group;The protection effects induced by the NMB0315 vaccines in BALB/c mice within 72 hours from high to low were as follows:the recombinant protein vaccine group, the combined immunization vaccine group, the nucleic acid vaccine group.
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The aim of this study is to investigate the distribution and sequence conservation of P6 outer membrane protein (OMP6)-encoding gene of nontypeable Haemophilus influenzae isolates as well as screen and identify the predominant T-and B-cell (T-B)-combined antigenic epitopes in OMP6 sequences and their immunogenicity.The entire omp6 genes of NTHi isolates were amplified by PCR and the amplification products were sequenced after T-A cloning.By using bioinformatic softwares,the sequence conservation and membrane location of OMP6 were analyzed as well as the T-B-combined antigenic epitopes in OMP6 were predicted.The immunogenicity and immunoreactivity of T-B-combined antigenic epitope peptides displayed by recombinant phage PⅢ proteins (rPⅢ) were determined by Western Blot assay and ELISA.The PCR showed that all the 35 NTHi isolates tested were detectable for omp6 gene.The identities of nucleotide and amino acid sequences of omp6 genes from 28 strains in the NTHi isolates were 98.3％-100％ and 99.3 ％-100％,respectively.OMP6 of NTHi was predicted as an outer membrane superficial protein that contains OMP6-2-25,OMP6-61-86 and OMP6-98-126 predicted T-B-combined antigenic epitopes.The immunoblotting assay and ELISA confirmed that OMP6-2-25 presented stronger hybridization band with NTHi antisera while 96.9％ (59/62),69.4％ (43/62) and 74.2％ (46/62) of serum samples from NTHi-infected children were positive for OMP6-2-25,OMP6-61-86 and OMP6-98-126 T-B-combined antigenic epitope peptides,respectively.All the results lead to a conclusion thatomp6 is an extensive distribution and sequence conserved gene of NTHi,and OMP6-2-25 is the predominant T-B-combined antigenic epitopes which can be used as the candidates for developing multiple antigenic peptide vaccine against NTHi.
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Objective To investigate the serovar distribution of Chlamydia trachomatis (Ct) isolated from male patients with urethritis in sexually transmitted disease (STD) clinic.Methods Urine specimens were collected from male patients with urethritis in STD clinic at Hospital of Dermatology,Chinese Academy of Medical Sciences between January 2013 and December 2013.Fluorescence-based quantitative PCR was performed to detect Ct DNA in these specimens.DNA was extracted from Ct-positive urine specimens,and nested PCR was conducted to amplify the VS1-VS2 regions of the outer membrane protein A (ompA) gene,followed by gene sequencing.The resulting sequences were aligned to reference sequences by the DNAStar5.0 software to determine Ct serovars.Results A total of 432 urine specimens were collected,and 33.1％ (143/432) of them were positive for Ct.The VS1-VS2 regions of the ompA gene were amplified from 127 out of the 143 Ct-positive specimens,but not from the other 16 specimens.Nine serovars were identified by gene sequencing among the 127 specimens,including serovar E (29 strains,22.83％),F (28 strains,22.05％),D (19 strains,14.96％),G (16 strains,12.60％),J (16 strains,12.60％),K (8 strains,6.30％),H (5 strains,3.94％),I (3 strains,2.36％) and B (3 strains,2.36％),and Ct serovars E,F,D,J and G accounted for 85.02％ among all the strains.Synonymous mutations were identified in 14 out of the 127 strains when compared with reference strains.Conclusions E,F,D and G serovars were the main Ct serovars in male patients with urethritis in STD clinic.The proportion of Ct serovar E strain was decreased,but that of serovar J strain was increased compared with 20 years ago.
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Objective:To construct NMB0315 eukaryotic expression recombinant vector ,detect specific humoral and cellular immune response induced by the recombint DNA vaccine intramuscularly in female BALB /c mice,evaluate the immunocompetence and immunoprotection of the vaccine , so as to provide experimental basis for the development of a novel nucleic acid vaccine against N.meningitidis serogroup B .Methods: The whole NMB0315 gene was amplified by PCR from the standard strains MC 58 genomic DNA,cloned into a plasmid pcDNA3.1(+),identified by double digestion of the recombinant plasmid with restriction enzymes and se -quencing.The recombinant vector pcDNA 3.1 (+)/NMB0315 was transfected into eukaryotic COS-7 cells and RAW264.7 cells, the NMB0315 protein was detected by immunocytochemical method and Western blot respectively .The levels of specific humoral and cellular immune response were detected after inoculating in female BALB /c mice intramuscularly with the recombinant plasmid .The immune protective effect was investigated with the DNA vaccine and the bactericidal titer of the immune serum was deter mined by serum bactericidal assay ( SBA ) in vitro.Results: The recombinant pcDNA3.1 (+)/NMB0315 was effectively transcripted and expressed in eukaryotic cells and the specific humoral and cellular immune responses were induced in the inoculated mice .In the re-combinant pcDNA3.1(+)/NMB0315 group ,the levels of serum IgG,IgG1,IgG2a,IgG2b and IgG3 and genital tract sIgA were significantly higher than in controls ( P<0.001 ) .The stimulation index in the culture supernatant of the spleen lymphocytes of the vaccine group was higher than that of the control group (P<0.05).The ratios of serum IgG2a/IgG1 in the DNA vaccine group were less than 1.The bactericidal titer of the NMB 0315+CpG group reached 1:128 following three immunizations , the protection rate of the vaccine group was 70%against the N.meningitidis strain MC58.Conclusion:The NMB0315 nucleic acid vaccine could induce higher levels of humoral immunity and cellular immunity and showed effective protection against N .meningitidis serogroup B , the immune serum had strong bactericidal activity in vitro .
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ObjectiveTo study the characteristics of the distribution and drug resistance ofAcinetobacter baumannii, and the epidemiology of the main strains among wards and hospitals, and to investigate the role of outer membrane protein in producing resistance against carbapenems.Methods 145Acinetobacter baumannii strains were collected from July 2013 to July 2014 from Huangdao Hospital Affiliated to Qingdao University and 401st Army Hospital. Antimicrobial susceptibility test was carried out with K-B disk diffusion method. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) was used for DNA typing and test of homology. The carO gene of outer membrane protein was amplified by PCR, and the outer membrane proteins were extracted by ultrasonication and ultracentrifuge method from 30 randomly selected carbapenem-resistantAcinetobacter baumannii and 17 carbapenem-sensitive strains. Sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) was used to analyze the expressions of outer membrane proteins.Results 145Acinetobacter baumannii strains were generally resistant to 16 common antimicrobial agents, with the highest susceptibility rate of 79.3% for minocycline, followed by susceptibility rate of 40.7% for amikacin. There were 128 carbapenem-resistant strains (resistance rate of 88.3%), 137 multidrug-resistant strains and 126 extensively drug-resistant strains. The detection rates of carO gene were 97.7%(125/128)and 17.6%(3/17) in carbapenem-resistant and sensitive strains respectively. Around position of relative molecular mass 47 000, 16 of 17 sensitive isolates were expressed this protein, while 20 of 30 resistant ones were detected nothing or fade; 13 of 17 sensitive isolates were expressed around position of relative molecular mass 37 000 and 29 000 while 25 were detected nothing or fade around position of relative molecular mass 37 000 and 23 were detected nothing or fade around position of relative molecular mass 29 000 in 30 resistant ones. 145Acinetobacter baumannii were classified into 8 types based on ERIC-PCR electrophoresis patterns, and the major prevalence types were type A (71 strains) and type E (37 strains).Conclusions Drug resistance of clinically isolatedAcinetobacter baumannii is a serious problem in two hospitals; drug-resistant strains are spread and epidemic among wards and hospitals. The carO gene of outer membrane protein is widespread in carbapenem-resistantAcinetobacter baumannii. The loss or fading of outer membrane protein may play an important role inAcinetobacter baumannii resistance to carbapenems drugs.
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Objective To screen and identify the predominant T-and B-cell ( T-B) combined an-tigenic epitopes on the outer membrane protein Loa22 of pathogenic Leptospira interrogans ( L.interrogans) stains and to further analyze their immunogenicity.Methods PCR analysis was used to detect loa22 gene in L.interrogans strains belonging to eight different serogroups or serovars prevalent in China.The PCR prod-ucts were sequenced after T-A cloning.A prokaryotic expression system for loa22 gene of L.interrogans sero-group Icterohaemorrhagiae serovar Lai strain Lai was constructed.The expressed recombinant protein rLoa22 was extracted by Ni-NTA affinity chromatography.The rabbit anti-rLoa22 serum samples and IgG were pre-pared.The T-B combined antigenic epitopes on Loa22 protein were predicted by using professional bioinfor-matic softwares.Phage display in combination with Western blot assay and ELISA were performed to identify the immunogenicity of the recombinant phage PⅢ protein-displayed and artificially-synthesized T-B com-bined antigenic epitopes, respectively.MTS assay and ELISA were performed to detect the activation of T cells and the expression of IL-2, IL-4 and IFN-γinduced by T-B combined antigenic epitopes, respectively. Results All of the tested pathogenic Leptospira strains were positive for loa22 gene, sharing 85.5%-99.8%homologies in nucleotide sequences and 93.9%-99.5%homologies in amino acid sequences.The construc-ted prokaryotic expression system for loa22 gene efficiently expressed the rLoa22 protein.Among four T-B combined antigenic epitopes (Loa22-77, Loa22-90, Loa22-125 and Loa22-157), only Loa22-90 epitope presented a strong positive band in Western blot analysis.The proliferation of CD4+T cells and the expres-sion of IL-2 ( Th1 ) and IL-4 ( Th2 ) were significantly enhanced by the stimulation with Loa22-90 epitope peptide (P<0.05).Conclusion Loa22 protein is a sequence-conserved genus-specific outer membrane protein of L.interrogans.The Loa22-90 epitope is the predominant T-B combined antigenic epitope of Loa22 protein, which might be used as a candidate antigenic epitope in the development of multiple antigenic pep-tide ( MAP) vaccines against Leptospira infection.
ABSTRACT
Background & objectives: Meningitis caused by Neisseria meningitidis is a fatal disease. Meningococcal meningitis is an endemic disease in Delhi and irregular pattern of outbreaks has been reported in India. All these outbreaks were associated with serogroup A. Detailed molecular characterization of N. meningitidis is required for the management of this fatal disease. In this study, we characterized antigenic diversity of surface exposed outer membrane protein (OMP) FetA antigen of N. meningitidis serogroup A isolates obtained from cases of invasive meningococcal meningitis in Delhi, India. Methods: Eight isolates of N. meningitidis were collected from cerebrospinal fluid during October 2008 to May 2011 from occasional cases of meningococcal meningitis. Seven isolates were from outbreaks of meningococcal meningitis in 2005-2006 in Delhi and its adjoining areas. These were subjected to molecular typing of fetA gene, an outer membrane protein gene. Results: All 15 N. meningitides isolates studied were serogroup A. This surface exposed porin is putatively under immune pressure. Hence as a part of molecular characterization, genotyping was carried out to find out the diversity in outer membrane protein (FetA) gene among the circulating isolates of N. meningitidis. All 15 isolates proved to be of the same existing allele type of FetA variable region (VR) when matched with global database. The allele found was F3-1 for all the isolates. Interpretation & conclusions: There was no diversity reported in the outer membrane protein FetA in the present study and hence this protein appeared to be a stable molecule. More studies on molecular characterization of FetA antigen are required from different serogroups circulating in different parts of the world.