ABSTRACT
<p><b>BACKGROUND</b>To express recombinant non-structural protein 3 of hepatitis C virus (HCV) in E. coli.</p><p><b>METHODS</b>The non-structural 3 (NS3) region DNA fragment of HCV was amplified by polymerase chain reaction (PCR) and inserted into inducible proeukaryotic expressive vector pET 30C(+)at Bam H1/EcoR1 sites. The competent BL21 (DE3) E.coli was transformed, and then cultured and induced with IPTG. The expressed HCV NS3 protein was confirmed with ELISA and dot blot hybridization using HCV NS3-specific single chain Fv (ScFv) antibody.</p><p><b>RESULTS</b>1 893 bp DNA fragment of HCV NS3 coding region was amplified by PCR technique. HCV NS3 expressive vector pET-NS3 was constructed. After transformation with pET-NS3 and induction with IPTG, recombinant HCV NS3 protein was expressed and confirmed by specific ELISA and dot blot hybridization.</p><p><b>CONCLUSIONS</b>The recombinant HCV NS3 can be expressed in E. coli.</p>
Subject(s)
Escherichia coli , Genetics , Gene Expression , Hepatitis C Antibodies , Genetics , Polymerase Chain Reaction , Recombinant Proteins , Genetics , Viral Nonstructural Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To develop a bacteria expression system to produce the fusion protein of humanized anti-HBsAg scFV and interferon-alpha.</p><p><b>METHODS</b>The expression vector was constructed after cleaving the plasmids harboring the humanized anti-HBsAg scFv and interferon alpha respectively and ligating to linearized pET22b subsequence. The expression of fusion protein in E.coli was analyzed by SDS-PAGE. The binding activity and antiviral activity of the fusion protein was characterized by competing inhibition test and cytopathic effect reduction.</p><p><b>RESULTS</b>The plasmid harboring the in frame arranged fusion gene was constructed and identified. After induction for 12h, a new band close to 4.5 10(4) was observed using SDS-PAGE. Results of competing ELISA and cytopathic effect reduction showed the fusion protein retained its specific binding activity and antiviral activities.</p><p><b>CONCLUSIONS</b>The construction and expression of the fusion gene of humanized anti-HBsAg scFv and interferon in E.coli are successful.</p>
Subject(s)
Humans , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetics , Gene Expression , Hepatitis B Antibodies , Genetics , Allergy and Immunology , Hepatitis B Surface Antigens , Allergy and Immunology , Hepatitis B virus , Immunoglobulin Fragments , Genetics , Allergy and Immunology , Interferon-alpha , Genetics , Pharmacology , Recombinant Fusion Proteins , Genetics , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To clone the unknown gene of hepatocyte protein interacting with hepatitis C virus core protein.</p><p><b>METHODS</b>Using the yeast dual hybrid system 3, bait plasmids of hepatitis C virus core were constructed. After identifying hepatitis C virus core protein that could stably expressed in AH109 yeast strains, we performed yeast two hybrid by mating AH109 with Y187 that transformed with liver cDNA library plasmids pACT2 and then plated on quadrople dropout (QDO) medium and assayed for alpha-gal activity. The genes of yeast colonies that could grow on QDO and had alpha-gal activity were sequenced.</p><p><b>RESULTS</b>Among the 30 positive colonies, we blasted the gene of the sixth colony; we coined human hepatitis C virus binding protein 6(Hu Hcbp6) with Genbank, realized that the Hu Hcbp6 shares as much as 98% homology with two cDNA without knowing functions. We have proved that Hu Hcbp6 could interact with hepatitis C virus core protein.</p><p><b>CONCLUSIONS</b>Hepatitis C virus core binding protein (Hu Hcbp 6 Genbank number: AY032594) was successfully cloned and identified. The study partly paved the way for investigating physiological function of the Hu Hcbp6.</p>
Subject(s)
Humans , Cloning, Molecular , DNA, Complementary , Genetics , DNA-Binding Proteins , Genetics , Hepacivirus , Molecular Sequence Data , Plasmids , Sequence Analysis, DNA , Transfection , Two-Hybrid System Techniques , Viral Core Proteins , Genetics , Metabolism , Yeasts , GeneticsABSTRACT
The aim of this study is to determine whether seeded Dacron with autologous venous tissue fragments shows significant 6-keto-prostglandin F 1? (6 K PGF 1? )production after in vivo implantation.The Dacron grafts seeded with autologous venous fragments were implanted into the inferior vene cava(IVC)of the 13 canines as seeded group;and the control group (8 canines),in which grafts were only preclotted with fresh blood. Plasma 6 K PGF 1? and TXB 2 were assessed at different time.All of the specimens explanted at exsanguination were observed with light microscopy and sanning electron microscopy and the amounts of 6 K PGF 1? from the luminal surface were assessed using ridioimmunoassay. The results showed that the total patency rate of the explanted vessels was higher in seeded group (61.5%) than that in control group ( 25% ),and that endothelial cells lined the whole luminal surface of Dacron at the 14th day after operation in seeded group,but thrombus covered the surface of Dacron at the 14th day in control ones. The level of 6 K PGF 1? from plasma and the luminal surface of Dacron grafts in seeded group was higher,but the level of plasma TXB 2 was lower than that in control group. It can be concluded that seeding Dacron with autologous venous fragments makes a new endothelial lining possible at two weeks,and can release more 6 K PGF 1? and thus lead to an improved patency rate of canine IVC reconstructions,so the graft patency might be determined according to the changes of plasma 6 K PGF 1? and TXB 2 level.
ABSTRACT
From 1978 to 1985, 14 patients with accidental injury of extrahepatic bile ducts at cholecystecto-my were reported. The incidence was 0.12%. The injury caused suffering, severe after effects for the patients.In the analysis of the causes, half of the cases occurred due to over-confidence and negligence of operating surgeons. Other causes were as unsatisfactory anesthesia, improper operative incision, blind clamping for control of hemorrhage in the area of the bile ducts, difficult dissection of the Calot's triangle because of anatomical anomaly, fibrosis, inflammation or adhesion. The methods of the prevention were discussed.According to various types of injury, different therapy should be taken. Early intervention, Roux-Y jejunal reconstruction, judicious use of Y tube and prolonged dilatation of hilar strictures will improve the outlook. No mortality and malfunction occurred.