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Nowadays,with the sharply increasing morbidity and stable high mortality,tumor has become the greatest threaten for human health and life.After taking a view of preservations,diagnoses and treatments on tumor, oncologists considered the cure rate had not improved evidently in patients with advanced tumor in the past several decades even though the total cure rate and survival rate had increased.Facing the puzzle,people should evaluate original tumorigenic theories again.A review about it was presented here.
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<p><b>OBJECTIVE</b>To express and identify soluble liver antigen (SLA) and cytochrome P-450 (CYP 2D6).</p><p><b>METHODS</b>SLA cDNA and CYP 2D6 cDNA were obtained from human liver tissue poly (A)+RNA by RT-PCR. The cDNAs were inserted into fusion expression vector PQE-30 site of BamH I and Hind III. SLA and CYP 2D6 were identified by the SDS-PAGE and Western blot.</p><p><b>RESULTS</b>SDS-PAGE analysis showed that there was a very strong stained band at about 47 kd and 50 kd, respectively. The products could specifically band to anti-SLA or anti-CYP 2D6 autoantibodies.</p><p><b>CONCLUSIONS</b>The clone and expression of SLA and CYP 2D6 provide useful substances for the diagnosis and research of pathogenesis on autoimmune hepatitis.</p>
Subject(s)
Humans , Autoantigens , Genetics , Cytochrome P-450 Enzyme System , Genetics , DNA, Complementary , Genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetics , Gene Expression , Hepatitis, Autoimmune , Genetics , Liver , Metabolism , Recombinant Fusion Proteins , Genetics , MetabolismABSTRACT
Objective To investigate the best fermentation and induction models of the engineered Escherichia coli, in order to obtain the highest expression level of humanized anti HBsAg Fab. Methods The optimal condition governing the growth of the Escherichia coli, with the flask shaking method and the best fermentation and induction conditions to yield the highest production level were explored, and then E coli were grown in fermentor using the fed batch method following the same principle under flask shaking condition to ensure the best production. Results The data obtained from flask shaking conditions showed that when the induction procedure started the amount of anti HBsAg Fab would reach the highest level at mid log growth phase under the induction condition of 25℃ and 0 2% arabinose. Using the DO stat fed batch method, the OD 600 value of the culture would reach 55 2, which corresponded to 110g/L bacterial wet weight. The biological activity of Fab was proved to have well preserved. Conclusion We established the optimal production technic of HBsAg Fab, and lay a foundation to produce HBsAg Fab on a large scale
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Human FceR I a subunit extracellular domain was cloned and expressed with 2 different expressing systems and Dot blot was used to detect its biological activity to bind with IgE,providing reference for binding mechanism of human FceR I a subunit extracellular domain with IgE. It was shown that FceR I a subunit extracellular domain from pBAD/g I A expressing system could bind with IgE, but the one from PQE30 expressing system could not bind with IgE. It is suggested that FceR I a subunit extracellular domain alone is sufficient to bind with IgE without P and Y subunit. The proper space configuration and disulfide bond of FceR I a subunit is necessary for binding with IgE, but its glycosylation is unnecessary.
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Objective: To evaluate the targeting activity in the animal model with human hepatoma, the 131I-human antiHBsAg Fab radioimmunoimaging was explored. Methods: Radioimmunoimagings were taken on different intervals after injection of 131 I-human anti-HBsAg Fab to the nude mice and tissue distribution was measured. The human anti-HBsAg Fab was compared with the murine monoclonal antibodies. Results: The experimental group developed tumor positive images after 3 days of radio-labeled monoclonal antibodies injection, and the peak accumulation of radio-activity on the 5th day.Statistics indicated the tumor/liver ratio of the human anti-HBsAg Fab, murine monoclonal antibodies and the control groups were 5.4,4.0 and 0.9 respectively on the 7th day. Conclusions: Our results suggest that the 131 I-human anti-HB-sAg Fab has a considerable targeting activity, and provide an evidence that it can be used as a novel humanized carrer for targeting therapy of hepatoma.
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Objective Construct an expression vector for anti-HBs Fab fragment and IFN-? fusion protein, then transform it into E.coli Top10 in order to produce a targeting drug for hepatitis B. Methods Using PCR and molecular clone techniques, we amplify the gene fragment of IFN-? with corresponding endonuclease sites and artificial linker at 5′,3′ termini,then recombine it within the vector of anti-HBs Fab gene in correct endonuclease sites at 3′ termini of the Lc gene, choosing the positive clone with PCR and transform it into E.coli Top10,after the expression and purification of the fusion protein, we use ELISA and Dot-blot to verify the antigenicity and binding activity to HBsAg. Results We got fusion protein consisting Lc and IFN-?. It has the IFN-? activity and HBsAg affinity which is similar to anti-HBs Fab fragment. Conclusions The successful expression of the fusion protein with both HBsAg affinity and IFN-? activity make it possible for immunobiotherapy drug targeted to HBV.
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Objective:To show whether the Fas Ligand gene induces mast cells apoptosis.Methods:RT-PCR was used to amplify the gene of rat Fas ligand extracelluar domain and transmembrane domain and cloned it into eukaryotic expression plasmid pcDNA3.1.Transcent transfect RBL-2H3,the expression of Fas ligand RBL-2H3 was detected by RT-PCR、Western blot.The Annexin V FCM was used to detect the RBL-2H3 apoptosis after the transfection of Fas Ligand.Results:It is successful to obtain the gene of rat Fas Ligand extracellular domain and transmembrane segment,cloning it into pcDNA3.1,FasL was expressed on the surface of RBL-2H3 and it's supernatant after the transfection of pcDNA3.1/FasL.The cell start to be apoptosis.Conclusion:Our study reveals that Fas Ligand gene transfection in RBL-2H3 can effectively induced apoptosis.It is a promising strategy for Fas Ligand to be used in the therapy of allergic disease. [
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Objective To assess functional affinity of humanized anti-HBsAg Fab in order to provide theoretical basis for the radioimmunotherapy of liver cancer bearing HBV. Methods With cognizance of the advantages of solid phase method in time consumption and convenience, we assessed the functional affinity of engineered anti-HBsAg Fab with non-competitive ELISA method. After a series of trials affirming the best concentration and the best time of HBsAg to cover the plate and the proper binding time of Fab to HBsAg to reach an equilibrium, we plotted the OD standard curve of the binding reaction of Fab and HBsAg using four grades of concentration of HBsAg and a series of consistency of Fab. We got the consistency of Fab at OD50% through the standard curve, and then calculated the affinity by Law of Mass Action. Results The affinity of anti-HBsAg Fab is between 10 7 -10 8 M -1 , which was only smaller by 10 times than that of anti-HBsAg IgG. Conclusion The Fab which we constructed in our lab, can strongly bind to the target antigen, thus providing a theoretical basis for its clinical use.
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The purpose of this study was to compare the efficiency of 3 different methods in purifying engineered anti-HBsFab. Anti-HBsFab Supernatants containing anti-HBsFab were prepared by ultrasound lysis of host cells of anti-HBsFab positive clone. Anti-Fab-sepharose gel, streptococcal protein G-sepharose gel (Prot G gel) and Ni-NTA-agarose gel were used to purify Fabs according to the reagents protocols. Then the concentrations of purified proteins were determined. SDS-PAGE was used for the measurement of purified Fabs'purity. HBsAg based ELISA was chosen to determine the Fabs' bio-activity. The results indicated that Fab recovery of different gels in equal volume were different. The recovery of Ni-NTA GEL was greater than that of Prot G gel, and the recovery of Prot G gel was greater than that of anti-Fab gel. Purity of the Fab isolated by 3 different gels was confirmed by SDS-PAGE. The binding capacity of anti-HBs Fab to HBsAg of these three gels had no signifticant difference. Our data suggested that each method had its advantage and usage limitation, Ni-NTA method demonstrated much better performance in economy, management, and efficiency over the other two methods.