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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.
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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.
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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.
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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.
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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 .
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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.