"Construction and preliminary validation of a new ""Controllable"" recombinant adenovirus vector carrying human insulin gene" / 实用医学杂志

ABSTRACT

Objective To design and construct a controlled adenovirus vector in degradating by itself after induction for solving the problem of stimulating host immune and producing replication adenovirus and providing a secure exogenous gene vectors for clinical practice. Methods Based on the traditional adenovirus vector AdEasyTM system, we inserted the Cre gene which belongs to Cre-LoxP system into the downstream of Tet-On inducible expression system. Two LoxP sites were inserted into two sides of the shuttle plasmid′s right arm genome. Then, the full-length human insulin gene was inserted HindIII enzyme site. After the recombinant adenovirus infected the rat bone marrow-derived mesenchymal stem cells , fluorescent protein expression and insulin secretion were detected before or after induction by Dox. Results A new controlled recombinant adenovirus vector carrying human insulin gene was constructed successfully, and was named AdEasyN/INS. After the transfection of this new vector into QBI-293A cells and rat bone marrow mesenchymal stem cells , green fluorescent protein could be observed. After induction by Dox, both of the ratio of fluorescent cells/total cell and the levels of insulin significantly decreased. Conclusion Construction and preliminary validation of a controlledrecombinant adenovirus vector carrying human insulin gene is constructed successfully , it could infect rat bone marrow mesenchymal stem cells, and degradate by itself after Dox induction, realize the controllability of exogenous gene carrier.