The Construction and Expression Confirmation of JC Virus T Antigen Expression Plasmid in Gastric Mucosa / 中国医科大学学报

ABSTRACT

Objective To construct and confirm the JC virus(JCV) T antigen expression plasmid using mouse keratin 19 (K19) promoter specific for the gastric epithelial cells.Methods The Ndel site was mutated by FCR with Bell insertion at both sides.The DNA fragment digested by Bcl Ⅰ was ligated with the plasmid containing K19 promoter via Bam Ⅰ site.The DNA sequence was confirmed by restriction enzyme digestion and direct DNA sequencing.Cytokeratin 19 protein was examined to screen gastric carcinoma cell for transfection of K19-JCV T antigen expression plasmid by immunohistochemistry.The Western blot was employed to detect the JCV T antigen expression in the gastric carcinoma transfectant.Results K19-JCV T antigen expressing plasmid was successfully constructed.The ACS strongly expressed cytokeratin 19 protein and was selected for the transfection of K19-JCV T antigen expressing plasmid.JCV T antigen was positively expressed in the AGS transfectant.Conclusion The synonymous mutation and compatible ligation are useful in the plasmid construction.The methy lation of restriction enzyme should be considered.It is meaning for the transgenic animal model of gastric carcinoma to successfully construct the JC virus T antigen expression plasmid in gastric mucosa.