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Abstract: To express human-mouse chimeric IgA antibody directed against H5N1 virus, an anti-H5N1 chimeric IgA antibody gene was constructed by joining the light and heavy chain variable region genes and the corresponding signal peptide coding sequences of the anti-H5N1 mouse monoclonal antibody H5N1-HA with the coding sequences of the constant region of the human IgA2 heavy chain and Kappa chain respectively. Then the full-length chimeric light and heavy chain expressing plasmids pEF-IGHA9 and pEF-IGK9 were constructed and transfected into the CHO/dhfr cells. The chimeric IgA antibody expression was confirmed by ELISA, SDS-PAGE and Western blotting. The successful expression of this anti-H5N1 chimeric IgA may help to provide a stand for developing passive immunological agents for H5N1 virus infection prophylaxis.
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Animales , Cricetinae , Humanos , Ratones , Anticuerpos Monoclonales , Anticuerpos Antivirales , Genética , Células CHO , Quimerismo , Cricetulus , Inmunoglobulina A , Genética , Alergia e Inmunología , Subtipo H5N1 del Virus de la Influenza A , Genética , Alergia e Inmunología , Proteínas Recombinantes de Fusión , Genética , Alergia e InmunologíaRESUMEN
Objective To investigate the effects of glutamate receptor signaling on melanoma cell dendrite morphology and cytoskeleton protein. Methods A metastatic human malignant melanoma cell line WM451LU was cultured and transfected by recombinant adenovirus vector carrying a cDNA encoding microtubule-associated protein 2a (MAP2a). MK-801, an antagonist of N-methyl-D-aspartate receptor (NMDAR), and CPCCOEt, an antagonist of metabotropie glutamate receptor 1 (mGluR1), were used to treat transfected or untrausfeeted WM451LU cells. Confocal microscopy and three dimensional atomic force microscopy were used to assess subcellular location of NMDAR2A, mGluR1 and MAP2a as well as the dis-tribution of α-tubulin in and dendrite morphology of WM451LU cells. The proliferation of WM451LU cells was estimated by cell survival growth curve. Results Confocal laser microscopy revealed that NMDAR2A, mGluRl and MAP2a were mainly co-localized in melanoma cell dendrites. Both MK-801 and CPCCOEt increased the density of microtubules in cell dendrites and dendritic branching of WM451LU cells, and both effects of MK-801 and CPCCOEt were enhanced by the expression of MAP2a. Furthermore, the proliferation of WM451LU cells was significantly inhibited by MK-801 of 100 μmol/L and CPCCOEt of 10 μmol/L. Conclusions In melanoma cells, glutamate receptors may participate in the development of dendrites, and anta- gonists of glutamate receptors could inhibit the proliferation of melanoma cells.
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Objective To obtain Chinese hamster ovary (CHO) cell lines that stably express a targeting complement inhibitor CR2-CD59.Methods The recombinant plasmid PEE14.1-CR2-CD59 was constru-cted by cloning the DNA fragment CR2-CD59 into plasmid PEE14.1,and the obtained plasmid was transfected into CHO cells by FuGENE 6.The clones with stable high expression of target fragment were selected by methionine sulfoximine (MSX),the expression of CR2-CD59 was analyzed by ELISA,SDS-PAGE and Western blotting analysis.Results Several stable expression clones were obtained,and CR2-CD59 was highly expressed in the secret form in CHO cells.SDS-PAGE analysis showed that the molecular weight of the recombined protein CR2-CD59 was consistent with the predicted one.ELISA and Western blotting results revealed that the CR2-CD59 could react with both anti-human CR2 and anti-human CD59 polyclonal antibodies.Compared with serum-containing medium,the protein was highly expressed in serum-free medium (P
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The thermogravimetric analysis was developed for the identification of powders and extracts of Radix Ginseng and Radix Panacis Quinquefolii. This method is simple,quick and accurate with small sample. It is avaliable for the identification of true and false product series of Radix Ginseng and Radix Panacis Quinquefolii in current market.