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Objective To clone the aldehyde dehydrogenase (adhA) gene from Methylovorus glucosotrophus and study its expression,purification and enzymatic characteristics.Methods The adhA gene was amplified and cloned to the expression vector pTIG.The AdhA was successfully expressed with induction in Escherichia coli BL21(DE3).The enzymatic characteristics were investigated by AHMT,and AdhA was purified by Ni+ exchange chromatography.Results AdhA accounted for more than 50% of the total cell proteins,and the purity was about 95%.With methanol as the substrate,the optimal pH of AdhA was 7.0,while the optimal temperature was 30℃.The enzymatic activity of purified AdhA remained about 60% when stored at room temperature for 6 days.Conclusion AdhA from MP688 is expressed in vitro,and methanol is the optimal substrate among all the substrates investigated.
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Objective To prepare and characterize specific monoclonal antibodies( McAbs) against the heavy chain of botulinum neurotoxin serotype A ( BoNT/AHc ) .Methods BALB/c mice were immunized with purified BoNT/AHc protein.After the fusion of mouse splenic cells with SP2/0 cells, hybridoma cell lines secreted McAbs against BoNT/AHc. The McAbs obtained were characterized by indirect ELISA, Western blotting and rapid isotypingassay before being used in ELISA to detect interaction sites in McAbs and BoNT/AHc preliminarily.Results Antigen protein BoNT/AHc of high purification was obtained.Four hybridoma cell lines secreting McAbs against BoNT/AHc were screened,named 1A4,3H3, 3H7 and 5H8,respectively.Their titers of McAbs were all above 3.0 ×103 .They were specifically combined with BoNT/AHc protein by Western blotting.The isotype of 1A4 and 3H7 was IgG1(Κ),that of 3H3 was IgM(Κ),and that of 5H8 was IgG2b(Κ).Additive ELISA showed that epitopes recognized by the four McAbs were close.ELISA analysis confirmed the interaction epitopes in McAbs and BoNT/AHc.Conclusion Monoclonal antibodies against BoNT/AHc are prepared and characterized,providing effective tools for studying the neutralizing antibody and antibody epitopes of BoNT/AHc.
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Objective To explore the clinical efficacy of PRGD composite nerve conduit in the treatment of human large-diameter,critical peripheral nerve defect in upper extremity.Methods From December,2011 to August,2014,19 patients with large-diameter,critical peripheral nerve defect in upper extremity were treated with PRGD composite nerve conduit.The patients were followeded-up periodically.The sensory and motor function recovery,high frequency ultrasound,and EMG were employed to assess the efficacy.Results The patients were followed up for an average time of 12-32 months(mean 21.75 ± 6.86 months),sensory and motor function recovered excellent in 7 patients,satisfactory in 7 patients,tolerable in 3 patients and no improvement in 2 patients were obtained according to the peripheral nerve function assessment standard built by British medical research council,the rate excellent and satisfactory results was 73.7％.Conclusion It is clinically promising to use PRGD composite nerve conduit to repair large-diameter,critical peripheral nerve defect in upper extremity,thus laying a foundation for its further application in clinical practice.
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BACKGROUND:Bone marrow mesenchymal stem cells (BMSCs) can be induced to differentiate into neuron-like cells directional y. Accordingly, BMSCs can be used as seed cells theoretical y in constructing tissue-engineered peripheral nerves. OBJECTIVE:Using combination of two cytokines to induce BMSCs differentiating into neuron-like cells directional y, and further to discusse its application in peripheral nerve injury. METHODS:BMSCs were isolated and purificated from the bone marrow of Wistar rats by using the differential adherence method. Basic fibroblast growth factor and epidermal growth factor were used to induce the BMSCs differentiating into neuron-like cells. The morphological change was observed and the neuronal specific markers were detected by immunohistochemistry technique. The morphological and immunohistological changes were also studied after the induce agent were removed. RESULTS AND CONCLUSION:With presence of morphological and immunohistochemical features of nerve cells induced by neurotrophic factors, BMSCs exhibited two or more processes that were interconnected as a meshwork;cellnucleus and nucleus could be observed with strong light refraction of cytoplasm. After immunohistochemical staining, neuroln specific enolase, neurofilament protein and synaptophysin protein positive cells were detected. A great amount of cells reversed to their original fibroblast-like morphology, and the expression of the three above-mentioned proteins decreased as the induce agent withdrawn. Our study showed that BMSCs can be induced to differentiate into neuron-like cells, but the transdifferentiation is a short-time reversible phenomenon.
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Objective To clone 2-hydroxyacid dehydrogenase (HADH) gene from Ketogulonigenium vulgare(KGV) Y25 and investigate its expression , purification, and enzymatic characterics .Methods The hadh gene was amplified from ketogulonigenium vulgare Y 25 and cloned into the expression plasmid pITG .The recombinant plasmid was transformed into Escherichia coli BL21(DE3).HADH was then successfully expressed with induction .To explore its enzymatic characteris-tics,HADH was purified by Ni +exchange chromatography .Results HADH constituted more than 50% of the total cell proteins analyzed by SDS-PAGE,with a relative molecular mass of about 35 ×103.With 2-keto-gulonic acid(2-KGA) as substrate, the optimal pH of HADH was at 8.0 ,while the optimal temperature of the purified HADH was at 45℃.Mean-while, such metal ions and chelating agents as Cu 2+, Ca2+, Mg2+, EDTA, and DEPC exerted little effect on enzymatic activities.The maximum initial activity of the enzyme towards 2-KGA reached 27 U/mg, and the Km was calculated as 2.6 mmol/L.The results of in vivo enzyme activity assay showed that HADH could metabolize 2-KGA.Conclusion The HADH gene form Y25 is successfully expressed in E.coli BL21 ( DE3 ) and the enzymatic characteristics of HADH are explored, which will facilitate subsequent studies on sorbose metabolic pathways and sugar acid conversion .