[Construction and expression of recombinant adenovirus vector carrying human endostatin, K5 and endostatin-K5 gene].

Aim: To construct the recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene respectively, and study their bioactivity in vitro.

Methods: Human endostatin, K5 and endostatin-K5 gene were amplified by PCR, which were then subcloned into shuttle vector pAd5-CMV-H1H2-MCS-6His by enzyme and ligation respectively. The positive recombinant plasmids linearized by Pac I were cotransfected into HEK 293 cells with the Pac I linearized adenoviral backbone plasmid using calcium phosphate precipitation method.

Genetically modified adenoviral vector with the protein transduction domain of Tat improves gene transfer to CAR-deficient cells.

The transduction efficiency of Ad (adenovirus) depends, to some extent, on the expression level of CAR (coxsackievirus and Ad receptor) of a target cell. The low level of CAR on the cell surface is a potential barrier to efficient gene transfer. To overcome this problem, PTD.

[Construction of an expression vector for high expression of siRNA].

Aim: To construct the vector for efficient expression of siRNA using pre-mir30 backbone.

Methods: By chemical synthesis method, pre-mir30 backbone introduced an appropriate restriction enzyme site for foreign shRNA inserting was cloned into an expressing vector containing U6 promoter. The silencing efficiency of a new siRNA expressing vector was detected by transfection and Western blot.