ABSTRACT
Objective To construct prokaryotic expression vectors for glutamate dehydrogenase(GDH)of Clostridium difficile(CD),and express recombinant GDH in Escherichia coli,and identify its antigenicityed.Methods The entire gene of GDH was cloned from ATCC43255 genome DNA.The recombinant antigens were expressed in E.coli with IPTG induction and purified by Ni-NATBeads.The antigenicity was detected using CD Qick Chek Complete dual-antigen EIA. Results Prokaryotic expression vectors of CD GDH were constructed successfully.The antigen could be identified by specific anti-GDH antibodies.Conclusion The GDH antigen can be used to prepare corresponding antibodies,which facilitate the development of immunoassay for CD GDH.
ABSTRACT
Objective To clone and express Mycobacterium tuberculosis fusion antigen ESAT-6 and CFP-10 ( EC) and to evaluate the biological activity of the fusion antigen EC in inducing specific cytokines secretion from THP -1 cells. Methods The fusion antigen EC gene was cloned into pET-30 a prokaryotic expression vector and expressed highly in E.coli BL21.Then, the THP-1 cells were stimulated with purified fusion antigen EC of different concentrations (10 and 20 μg/ml).Culture supernatants were collected after 12 h and 24 h, respectively.The secretion levels of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-αand IFN-γin THP-1 cell culture supernatants were detected using Bio-PlexProTM Assays kit.Results The M.tuberculosis fusion antigen EC was cloned and expressed successfully .The secretion levels of IL-6, IL-8 and TNF-αin EC infected THP-1 cells were significantly higher than those in THP-1 cells (P<0.05).The secretion levels of other cytokines did not change significantly .Conclusion The obtained M.tuberculosis fusion antigen EC has biological activity in inducing the THP-1 cells to secrete a higher level of IL-6,IL-8 and TNF-α.
ABSTRACT
Objective To compare sensitive difference of docetaxel between the triple negative breast cancer(TNBC)cell line CAL-51 and non TNBC line T47D and analyze mechanisms underlying docetaxel resistance in former cells. Methods Cell activi?ty was determined by MTT method and IC50 value was calculated;Wright-Giemsa stain was used to analyze the effect of docetaxel in the morphology of CAL-51 and T47D cell lines. Flow cytometry(FCM)was performed to determine cell cycle distribution and apoptosis. Realtime fluorescence quantitative PCR was used to compare the relative gene expression levels.The anti-apoptosis protein Bcl-2 and caspase family protein expression levels were determined by Western blot. Results Wright-Giemsa stain showed significant morpholo?gy change in T47D cells by docetaxel treatment. Further flow cytometry results confirmed that docetaxel could significantly induce apoptosis in T47D cells compared to CAL-51 cells(P<0.01). The result of realtime fluorescence quantitative PCR revealed that anti-apoptosis protein Bcl-2 was significantly higher expressed in CAL-51 cells(P<0.05). Immunoblot analysis revealed docetaxel treat?ment induced the instrinsic pathways in both CAL-51and T47D cells,but the activated pathway of executioner caspase was different. Conclusion Our present study shows that docetaxel induces different intrinsic apoptosis pathway in CAL-51 and T47D cell lines. An?ti-apoprosis protein Bcl-2 is highly expressed,which might be the underlying mechanism of docetaxel resistance in TNBC cell line-CAL-51.
ABSTRACT
Objective To analysis the positive rates of glutamic acid decarboxylase autoantibody (GADA)and zinc transporter 8 autoantibody (ZnT8A)in newly diagnosed type 2 diabetes patients.Methods GADA and ZnT8A were detected in 101 ca-ses of newly diagnosed type 2 diabetes mellitus patients using ELISA.Results The positive rate of GADA was 21.78%,the positive rate of ZnT8A was 17.82%,and the common positive rate of GADA and ZnT8A was 8.91%.There were no corre-lations between GADA or ZnT8A autoantibodies and the patient’s sex (t=-0.724,-0.550;0.903,1.359,P >0.05),age (t=-0.724,-0.550;0.903,1.359,P >0.05),blood glucose (r=0.290,0.110;-0.264,-0.047,P >0.05),cholesterol (r=-0.047,0.004;0.154,-0.138,P >0.05),triglyceride (r=-0.092,-0.054;-0.217,-0.023,P >0.05),and low density lipoprotein (r= - 0.045,- 0.027;0.202,- 0.025,P > 0.05).Conclusion It should be screened autoantibodies timely for newly diagnosed type 2 diabetic patients in order to diagnosis the Latent autoimmune diabetes in adults early.
ABSTRACT
Objective To obtain different fragments of human carboxypeptidase H,and evaluate the diagnostic application of the recombination carboxypeptidase H in detecting autoantibody.Methods The coding gene of carboxypeptidase H was ob-tained by RT-PCR.The corresponding prokaryotic expression vectors were constructed and transformed into E.coli to in-duce the expression of the recombination different fragments of carboxypeptidase H.Using these antigen fragments as the coating antigens,the enzyme-linked immunosorbent assay (ELISA)was established for the detection of carboxypeptidase H autoantibody in 95 newly diagnosed type 2 diabetes patients.Results Three fragments of human carboxypeptidase H were obtained,in which the 42~476aa fragment antigen was ideal one.Using the full-length carboxypeptidase H as coating anti-gen,the positive rate of carboxypeptidase H autoantibody was 8.42%.Conclusion Because of the favorable antigenicity,the 42~476aa fragment antigen of carboxypeptidase H could be the candidate antigen for discrimination and diagnosis of latent autoimmune diabetes in adults.
ABSTRACT
Objective To establish the enzyme-linked immunosorbent assay (ELISA) for detecting human serum fumarate hydratase (FH) antibody and evaluate its role in the diagnosis of autoantigen in autoimmune hepatitis (AIH).Methods The indirect ELISA was established using FH protein,and the reaction conditions were determined.Then,the anti-FH antibody were detected in the serum of 88 AIH patients,56 primary biliary cirrhosis (PBC) patients,50 chronic hepatitis B (HBV) patients,36 chronic hepatitis C (HCV) patients and 98 healthy controls(HC).The results were analyzed with chi-quare and Kruskal-Wallis H methods.Results The ELISA for detecting human anti-FH antibody was established successfully and the optimal reaction conditions were defined.The positive rate of anti-FH antibody in the AIH group (40％) was significantly higher than HC (3％,x2=38.44,P＜0.01),PBC group (7％,x2=18.45,P＜0.01),CHB group (2％,x2=23.59,P＜0.01) and CHC group (6％,x2=14.29,P＜0.01).Anti-FH antibody which was used to diagnose AIH revealed a sensitivity of 40％ and specificity of 94％.Conclusion We have established the ELISA,which is used to detect human anti-FH antibody.It can be detected predominantly in AIH,and this implies that anti-FH antibody may be useful in improving the diagnosis of AIH.
ABSTRACT
Objective To develop an antigen retrieval method for detection of human mammaglobin ( hMAM) immuno-histochemcal staining in old paraffin-embedded specimens .Methods The tissue sections in test group were put into dis-tilled water after deparaffinization and then moved into citric acid buffer ( pH 3.5) for 10-15 min.The other two meth-ods,microwave method and high pressure cooker method ,were compared as control groups at the same time .Finally, immu-nohistochemistry SP method was used to check the antibody in the sections .Results The color appearance in the test group (pH 3.5 citric solution) was better than that of microwave oven and high pressure cooker groups .In the test group, tissue sections were not easily cast off from the slices .Conclusion In this study,we have established a new and simple antigen retrieval method which will contribute to immunohistochemistry technology .
ABSTRACT
Objective To provide the candidate antigens for immunological diagnosis by analyzing the expression of nu -cleoprotein ( NP) of Ebola virus. Methods BioSun software was used to predict the NP epitopes. The bridging-PCR was used to synthesize the NP gene. The pBVIL1 vector was used to clone and express the NP gene. Results The 360-739 aa of NP was confirmed to be the dominant antigen by BioSun software. The recombinant NP dominant antigen was expressed in E.coli with molecular weight of 58 ×103.The specificity of ELISA based on recombinant NP was 99.24% (130/131) in negative samples. Conclusions The dominant NP antigen can be potentially used for developing Ebola virus diagnostic reagent.
ABSTRACT
ObjectiveTo discuss the relationship between the cross-reactivity of antibody against the hypervariable region 1 ( HVR1 ) of hepatitis C virus and early viral response ( EVR ) in patients undergoing antiviral therapy.MethodsBy ELISA and HCV HVR1 antibody cross-reactivity matrix reagent,the differences of anti-HCV hypervariable region antibody were tested in baseline serum from 46 patients with chronic hepatitis C before antiviral therapy.HCV genotyping and HCV RNA were analyzed by RT-PCR method.The HCV RNA assay was done at three time points:before treatment,pegylated interferon in combination with ribavirin therapy for 12 and 48 weeks.ResultsIn 46 cases of chronic hepatitis C patients,there were 16 cases with HCV 2a type,30 cases with l b and 33 patients obtained EVR.The EVR incidence of type 2a[ 93.8％ ( 15/16 ) ] was higher than that of type 1 b[ 60.0％ (18/30),x2 =4.316,P ＜ 0.05 ].In the EVR group of type 1b chronic hepatitis C patients,the positive number of average multi-target HVR1 antigen was ( 12 ± 4),which was significantly higher than that in the Non-EVR patients [ (7 ± 5 ),t =2.797,P ＜0.01 ].Bv the Fisher exact test,it was showed that patients'serum response to HVR1 antigens numbered 001,003,009,013,016 were higher in EVR group than those in non-EVR group,with statistically significant (P ＜ 0.05 ).ConclusionThe cross-reactivity of HVR1 antibody may play an important role in predicting the effectiveness of antiviral therapy.
ABSTRACT
Objective To research CpG and Al(OH)3 adjuvants enhancing immunogenicity of hepatitis C virus(HCV) recombinant ptotein combined vaccine(TIE).Methods BALB/c mice were immunized with candidate vaccine TFE using CpG,Al(OH)3,Al(OH) 3 + CpG,or freund's adjuvant(FA) as the adjuvant.Five mice were sacrificed after 10 d of the last immunization.Specific antibodies in sera were tested by enzyme-linked immunosorbent assay(ELISA).Splenic cells were isolated and levels of IFN-γ,IL-4 and cytotoxic T lymphocyte(CTL) cytotoxicity assay were messuredin vitro.The remaining mice were subcutaneouly injected with 1 × 106 SP2/0-NS3 cells on the back to investigate the protective effects.The differences of means between groups were compared by LSD-t test.Results The specific CTL activity of TFE + A1(OH) 3 + CpG group was higher than TFE + FA group and TFE + CpG group(P ＜ 0.05).The level of IFN-γsecreting cells in TFE + Al(OH)3 + CpG group was higher than that in TFE + M(OH)3 group or TFE + CpG group(P ＜ 0.05).Conclusion Combining Al(OH) 3 and CpG could enhance specific cellular immunogenicity of candidate HCV vaccine TFE.TFE + M(OH) 3 + CpG could effetively prevent the attack of tumor cell SP2/0-NS3 expressing nonstructural protein NS3 of HCV.
ABSTRACT
Objective To investigate immune responses and protective effect induced by two recombinant proteins of hepatitis C virus(HCV)in BALB/c mice.Methods BALB/c mice were immunized with recombinant proteins HCV-T and(or)HCV-F4HVR1 three times.Specific antibodies in sera were tested by enzyme-linked immunosorbent assay(ELISA).Five mice were sacrificed after 14 days of the last immunization.Splenic cells were isolated and levels of interferon(IFN)-γ,interleukin(IL)-4 and cytotoxic T lymphocyte(CTL)cytotoxicity assay were measured in vitro.The remaining mice were subcutaneously injected with 1.0×106 SP2/0-NS3 cells on the back to investigate the protective effects.The differences of means between groups were compared by LSD-t test.Results Compared with phosphate bufter saline(PBS)group,combined immunization with HCV-T and HCV-F4HVR1 induced higher levels of specific IgG against HCV-F4HVR1(t=3.815,3.762,P＜0.05),HCV-NS3-specific CTL response(t=3.971,P＜0.05)and IL-4(t=3.512,3.417,P＜0.05)and IFN-γ(t=3.813,3.426,3.671,P＜0.05)secretions.Conclusion High levels of specific humoral immunity and cellular immunity are induced in vivo after combined immunization with HCV-T and HCV-F4 HVR1,which could effectively prevent from the attack of SP2/0-NS3 cells.
ABSTRACT
Objective To develop the technique to detect total core antigen of HCV(Total HCV-cAg) by Enzyme-Linked Immu-nosorbent Assay (ELISA) and apply it for clinical diagnosis. Methods 201 serum samples with anti-HCV antibody were detected total HCV-cAg after pre - treating the samples, then the sensitivity of results were compared with HCV RNA tests. Among them, 176 cases was determined by FQ-PCR, and 25 cases by RT-PCR for HCV-RNA. Results HCV RNA was found in sera from 88 of 201 samples (43.8%). Total HCV-cAg was positive in 71 (35.3%) of 201 samples . There was no significant difference between the detection rate of HCV RNA by PCR and total HCV-cAg by ELISA. Conclusion Detection of total core antigen of HCV is suitable to be used as to diagnose HCV in clinic.
ABSTRACT
Objective To design a complex hepatitis C vires(HCV)=E1 antigen,and to search its application in HCV vaccine and diagnosis test.Methods Through consulting the database and widely comparison of sequences from HCV E1 of different genotype,the representative immunodominant epitope sequences were selected from all the six genotypes.Their genes were deduced according to the preference codon in E.coli and three fragments were designed to contain the six epitopes.They were chemically synthesized and cloned into pBVIL1 vector separately.The cloned fragments were conjugated together profiting from pBVIL1's property,and then a doubled pan-DR helper T cell epitopes(PADRE)gene was inserted to form a complex expression plasmid.The transformed E.coli cells with this plasmid were cultured and induced to express recombinant protein and the antigenic activity of the product was tested.Results An expressing plasmid containing 8 epitopes from HCV genotype 1a,1b,2a,3a,4a,6a and a doubled PADRE sequences was constructed successfully and the engineering E.coli transformed with this plasmid highly expressed after inducing culture at 42℃ in an inclusion manner.The immunological activity of the purified recombinsnt multi-epitope antigen shows that:(1)It can react with a great part of sera from HCV positive patients by indirect ELISA.(2)It can induce notable specific humoral immunity in injected mice.Conclusion The novel constructed expressing plasmid and its product may be useful in study of a newly HCV vaccine as well as an antigen for HCV immunoassays.
ABSTRACT
Objective To investigate the cellular and humoral immune responses and protective effect induced by co-immunization with two multi-epitope combinant antigens. Methods Mice were co-im-munized with the muhi-epitope HCV-T and HCV-E1 antigens three times. Sera antibodies IgG, IgG1 and IgG2a were tested by ELISA. Spleens from BALB/c mice immunized were removed 10 days after the last im-munization. CTL activity was assessed using LDH cytotoxicity assay kit. IFN-γ- and IL-4-secreting cells were quantified using ELISPOT kit. Two weeks after the final immunization, the mice were challenged sub-cutaneously(s, c. ) at the back with 106 SP2/0-NS3 cells, and protective effect was observed. For therapy, 106 SP2/0-NS3 cells were implanted into the back of BALB/c mice. Seven days later, mice were immuniza-tion three times. Therapy effect was observed. Results Co-immunization with HCV-T and HCV-E1 induced high tiers of HCV-El-specific IgG, IgG1 and IgG2a antibodies, and high level of CTL activity. Synergistic effect in frequencies of both specific IFN-γ-secreting cells and IL-4-secreting cells was observed in mice co-immunized. Prophylactic as well as therapeutic administration of mT + mE1 in mice led to protecting mice against SP2/0-NS3 cells. These results suggested that mT + mE1 was potential as a prophylactic as well as therapeutic HCV vaccine. Conclusion Co-immunization with HCV-T + HCV-EI induced protective humor-al and cellular immune response. HCV-T + HCV-E1 was potential as a recombinant HCV vaccine.
ABSTRACT
Objective To study the hepatocyte cells infected by hepatitis C virus (HCV) positive serum. Methods Human hepatocyte 7701 was incubated with HCV RNA-positive and HCV antibody(Ab) negative sera BP52. Then, the expression of HCV antigen and the presence of HCV-RNA in cell and supernatant were assayed by RT-PCR, sequence analysis, immunofluorescent staining, Western blot, confocal laser microscopy. The ultrastructural changes of infected cells were observed by electro-microscopy. Results Plus-strand RNA and minus-strand RNA were intermittently detected in cell and/or supernatant on day 7-45 after infection. Sequence analysis demonstrated that the positive DNA nucleic acids were identified with HCV 5′-non-coding region(NCR) sequence. HCV core and NS3 protein were expressed in cytoplasm of infected cells. After 2 or 3 weeks, obvious intracellular ultrastructural changes and virus-like particles were observed. Conclusion human hepatocyte 7701 could support replication of HCV in vitro, which could be a useful tool for setting up cell model of HCV infection and studying the mechanism of HCV infection.
ABSTRACT
Objective To detect antibodies against the hypervariable region 1(HVR1) of hepatitis C virus in blood screening.Methods The HVR1 antibodies were detected by F4HVR1 antigen,and then were compared with other antibodies of HCV.Results Among HCV-RNA positive samples,HVR1 antibody was 96.8% positive.The positive rate of HVR1 antibodies in 90 suspected HCV-infected samples was 61.1%,which was close to those against C and NS3,and higher than NS4 and NS5(P
ABSTRACT
Objective To establish a sandwich enzyme-linked immunosorbent (ELISA) method for early diagnosis of hepatitis C. Methods Immunization of Balb/c mice with hepatitis C core antigen were prepared by genetic engineering. Mouse monoclonal antibodies (McAb) to anti-HCV-core Ag were obtained. A sandwich ELISA kit for detecting HCV-core Ag was developed by using four strains of anti-HCV-core Ag McAb. One hundred and twenty five serum specimens with increased ALT but negative for anti-HCV, anti-HIV, and anti-Tp tests were tested. Results Nine of the 125 specimens were positive for HCV-core Ag. Conclusion The double sandwich ELISA kits for detecting HCV-core Ag may be useful for the early diagnosis of hepatitis C .
ABSTRACT
With light and electron microscope, we studied the morphology of the thymus of the neonatal mouse. The results showed: 1.the lobules of the thymus had not well developed and there was no distinct demarcation between the cortex and medulla; 2.a cyst composed of epithelial cells with microvilli or cilia might be frequently seen in the medulla; 3.occasionally small lymphocytes with some glycogen particles in their cytoplasm were observed; 4.only a few small-sized thymic corpusles existed in the medulla, The article also described the ultrastructure of the lymphocytes, epithelial reticular cells, macrophage, interdigitating cell and blood-thymus barrier in the thymus of the neonatal mouse.
ABSTRACT
The ultrastructure of dissociated brain nerve cells from embryonic chicken cult ivated in vitro was studied both under SEM and TEM. Primitive synaptic junctions among the bipolar and multipolar neuronal cell bodies and its processes forming networks of fibers appeared after 4 days of culture. The presynaptic bags and desmosome-like cell junctions were observed simutaneously. Axo-dendritic, axo-somatic and axo-axonal synapses increased in number after 7 days of incubation onwards. Neuronal degeneration and proliferation of fibrous glial cells were evident in10 and 14 days cultures.