1. Construction and identification of polycistron adenoviral expression vector PCA13/FasL-IRES-iNOS
- Author
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Zhi Qi Zhang, Jiang Feng Qiu, Chun Xia Luo, Fu Rong Yu, and Zhi Yong Wu
- Subjects
Fas Ligand Protein ,Genes, Viral ,Genetic enhancement ,Genetic Vectors ,Nitric Oxide Synthase Type II ,chemical and pharmacologic phenomena ,Biology ,Fas ligand ,Viral vector ,Adenoviridae ,Plasmid ,Viral Structural Proteins ,Expression vector ,Membrane Glycoproteins ,fungi ,Gastroenterology ,Gene Transfer Techniques ,Genetic Therapy ,Molecular biology ,Internal ribosome entry site ,Transformation (genetics) ,Apoptosis ,Hypertension ,Nitric Oxide Synthase ,Ribosomes ,Plasmids - Abstract
The immunogenicity of adenovirus that causes the clearance by the host, is the major barrier to successful gene transfer. Some special cells such as cornea-epithelial cells and testicular Sertoli cells can induce apoptosis in immune cells with Fas molecule through expression and secretion of Fas ligand (FasL). Aims: To construct FasL and inducible nitric-oxide synthase (iNOS) co-expressed PCA_(13) plasmid used for packing of adenovirus, so as to observe the immunoprotective effect of FasL on adenovirus vector. Methods: By way of internal ribosome entry site (IRES), genetic engineering techniques as preparation and transformation of competent cells, plasmid extraction, agarose gel electrophoresis, restriction enzymolysis were used for the construction of polycistron adenoviral expression vector PCA_(13)/FasL-IRES-iNOS, which could co-express with FasL and iNOS gene after multi-subcloning steps. Results: FasL and iNOS gene connected with IRES were successfully cloned to PCA_(13) plasmid and verified by enzymolysis (600 bp FasL, 1000 bp IRES and 4000 bp iNOS), and the gene sequence was concordant with the Genebank. Conclusions: It is one of the crucial steps to construct successfully the polycistron adenoviral expression vector PCA_(13)/FasL-IRES-iNOS for the gene therapy of portal hypertension.
- Published
- 2004