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Objective To construct human EpCAM eukaryotic recombinant plasmid ,and to study the expression of EpCAM pro‐tein in HepG2 cells .Methods The recombinant plasmid ,named pcDNA3 .1(+ )‐EpCAMwas constructed by cloning EpCAM gene into eukaryotic vector pcDNA3 .1(+ ) .Then HepG2 cells were transfected with EpCAMrecombinant plasmid .The eukaryotic ex‐pression of EpCAM protein was verified by immunofluorescence ,Western Blotting and flow cytometry .Results The construction of EpCAM recombinant eukaryotic plasmid was identified by restriction enzyme digestion .The results of immunofluorescence , Western Blotting ,and flow cytometry consistently indicated that EpCAM protein were successfully expressed in HepG2 cells .Con‐clusion Human EpCAM eukaryotic vector is constructed and EpCAM protein could be expressed well in HepG2 cells ,which laid a foundation for further research on the function ofhepatocarcinoma cells highly expressing EpCAM .
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Objective To establish a chemiluminecentdetection method ( CLIA ) of HCV IgG antibody for the detection of HCV infection and therefore lay a foundation for the research and development of testing kit.Methods Based upon the indirect ELISA method, the microwell plate was coated with HCV-NS3 and HCV-Core antigen expressed through gene engineering, and the anti-human IgG antibody was labeled with horse radish peroxidase.In this way, the chemiluminesent detection method of HCV IgG antibody was established.Meanwhile, the serum specimen of randomly selected 198 patients infected with HCV from No.302 Hospital of PLA and 222 blood donors, and the results were compared.Results The HCV-IgG antibody, a positive consistent rate of 99.0%( 196/198 ) , a negative consistent rate of 98.2%(218/222), and a total consistent rate of 98.6%(414/420) were found through testing 420 serum specimen with self-made agent and contrast agent.One HCV positive serum was repetitively tested with the self-made agent for 10 times, and a coefficient of variation ( CV) of less than 10% was found.Conclusion The chemiluminescent detection method of HCV IgG antibody is initially established, and the method, with an outstanding specificity and sensitivity, is applicable for screening blood donors, clinically detecting HCV infection as well as epidemiological survey.
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Epithelial cellular adhesion molecule (EpCAM)is a transmembrane glycoprotein expressed in epithelial tissues and most epitheli-al-derived malignant tumors.EpCAMis associated with cell adhesion,migration,proliferation,and differentiation,as well as tumor devel-opment and progression.In recent years,EpCAMhas been identified as a stem cell marker of hepatocellular carcinoma (HCC).This paper elucidates the roles of EpCAMin the diagnosis,treatment,and prognosis of HCC,which shed light on the potential molecular targeted thera-py in future.
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Objective To understand the incidence and death patterns of viral hepatitis in China and provide evidence for the prevention and control of viral hepatitis.Methods The analysis was conducted on the incidence and death data of viral hepatitis published by National Health and Family Planning Commission during 2004-2013.Results The incidences of viral hepatitis in Guizhou,Yunnan,Tibet,Gansu,Qinghai,Ningxia and Xinjiang provinces (autonomous region) were high.The major forms were hepatitis B (80.63/100 000) and hepatitis C (9.68/100 000),accounting for 80.90％ and 9.25％ of the total reported viral hepatitis cases respectively.The incidences of hepatitis A and unidentified hepatitis decreased and the incidence of hepatitis B,C and E increased during this period.During the 10 years,10 008 deaths caused by viral hepatitis were reported (1 001 deaths per year).The reported deaths caused by hepatitis A,hepatitis E and unidentified hepatitis decreased during this period.The reported deaths caused by hepatitis B were in a downward trend,but the constituent in total cases remained high.The reported deaths caused by hepatitis C were in an upward trend.Conclusion During 2004-2013,the overall incidence of viral hepatitis showed no downward trend in China.The incidence of hepatitis B remained high,and the incidence of hepatitis C showed an obvious upward trend.The overall death rate and case fatality rate of viral hepatitis showed a downward trend,but hepatitis B remained the main cause of viral hepatitis related death,and the death caused by hepatitis C was in increase.Hepatitis B and hepatitis C are the major targets in the prevention and treatment of viral hepatitis in China,and the 7 western provinces (autonomous region) with high incidences are the key regions of the prevention and control.
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Objective To isolate and identify the causative agent in an incident with crowd fever of adults in Henan province.Methods The cells was inoculated by the throat swabs of the patients and followed by neutralization assay and other molecular methods.Then indirect immunofluorescence assay was performed to detect the specific IgM/IgG antibodies against virus in the serum of the patients.Results We have isolated 2 strain adenoviruses from 10 swab samples,which were both identified as Ad11 by sequence analysis and neutralization test.6 of 10 samples were positive fer IgM specific for adenovirus and 3 positive for IgG.The remaining sample was negative for both.Conclusion The causative agent in this incident with crowd fever of adults was Ad11 in group B.
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<p><b>OBJECTIVE</b>To describe and evaluate a double-antigen sandwich ELISA for detecting human immunodeficiency virus type 1/2 (HIV-1/2) specific antibodies.</p><p><b>METHODS</b>The peptides gp41.1(sp1), gp41.2(sp2), gp120(sp3) and p24(sp4) of HIV-1 and gp36(sp5) of HIV-2 were artificially synthesized. Then sp1, sp3, sp4 and sp5 were used as coating antigens; sp1, sp2, sp4 and sp5 labeled with HRP were used as conjugates in this sandwich ELISA.</p><p><b>RESULTS</b>The specificity and sensitivity of the assay were both 100% in detecting anti-HIV of 40 control sera of the second generation panel, higher than indirect ELISA (specificity 90% and sensitivity, 65%, respectively). All of 210 sera from individuals with other diseases were negative for anti-HIV. The consistency rate was 100% when our sandwich ELISA and Abbott HIVAB were used to detect anti-HIV in 90 healthy blood donors and 88 HIV infected individuals.</p><p><b>CONCLUSIONS</b>The results showed that this sandwich ELISA for detection of anti-HIV is specific, sensitive and convenient, and it is suitable for screening blood donors and detecting HIV infection.</p>
Subject(s)
Humans , Enzyme-Linked Immunosorbent Assay , Methods , HIV Antibodies , Blood , HIV Infections , Blood , Virology , HIV-1 , Allergy and Immunology , HIV-2 , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To screen human monoclonal Fab fragments against HIV-1 gp120 peptide binding chemokine receptor.</p><p><b>METHODS</b>A synthesized polypeptide containing 23 amino acid residues of the gp120 antigen epitope binding chemokine receptor was coated as the solid-phase antigen. After biopanning from the HIV-1 phage Fab antibody library, the acquired positive clones were tested and sequenced.</p><p><b>RESULTS</b>One clone of human phage Fab monoclonal antibody against HIV-1 gp120 polypeptide was acquired. It has high affinity, specificity and inhibition rate and it belongs to IgG I subclass and kappa type. Its Vh H and V kappa were derived from Vh III and V kappa III.</p><p><b>CONCLUSIONS</b>The human phage Fab fragment against HIV-1 gp120 antigen site binding chemokine receptor was acquired.</p>
Subject(s)
Humans , Amino Acid Sequence , Antibodies, Monoclonal , Genetics , Antibodies, Viral , Genetics , Bacteriophages , Genetics , Cloning, Molecular , HIV Envelope Protein gp120 , Immunoglobulin Fab Fragments , Genetics , In Vitro Techniques , Molecular Sequence Data , Peptide Library , Receptors, Chemokine , Metabolism , Receptors, HIV , Allergy and ImmunologyABSTRACT
Objective To study the changes in serum endotoxin level of the patients with primary biliary cirrhosis (PBC) and the relationship between endotoxin and serum biochemical parameters. Methods Clinical data of 63 cases PBC patients confirmed by liver puncture biopsy were retrospectively studied. The clinical features, results of laboratory tests, pathological findings of all the 63 patients were analyzed. Endotoxin level was determined by limulus amebocyte lysate test in the serum obtained from the 63 cases of patients with primary biliary cirrhosis, and then compared with that of 30 healthy individuals. Results The sex ratio (male to female) of PBC patients was 1 to 8, and the mean age was 43.8 years. The prevalent complaints were jaundice and fatigue. 70.3% patients showed AMA positive. The levels of ?-glutamyltransferase (r-GGT), alkaline phosphatase (ALP) and bilirubin in the serum were markedly elevated in the majority of the patients. Compared with the early stage, the levels of ALT and AST declined slightly during the advanced stage (P