Preparation and identification of recombinant adenoviral vectors containing human wild type SPK and its mutant gene
- VernacularTitle:携带人鞘氨醇激酶及突变体基因重组腺病毒载体的制备及表达
- Author:
Jun YI
;
Zhuozhuang LU
;
Haifen DUAN
- Publication Type:Journal Article
- Keywords:
adenoviridae;
sphingosine kinase;
mutant type
- From:
Medical Journal of Chinese People's Liberation Army
1981;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective In order to further understand the bioactivity of sphingosine kinase, high efficient replication defective recombinant adenovirus carrying the wild type SPK gene (SPK WT ) and SPK mutant gene (SPK DN ) were constructed respectively. Methods The SPK genes of interest were subcloned into a shuttle vector pshuttle cmv respectively. Pshuttle cmv SPK WT and Pshuttle cmv SPK DN were linearized by PmeI and transformed respectively into the competent cells of E. coli BJ AD 1 cells which contain adenoviral backbone plasmid pAdEasy 1. The linearized recombinant plasmids were transfected into adenovirus 293 packaging cells respectively,and then recombinant adenoviruses were harvested. We performed the PCR, Western blot and enzyme assays to identify the recombinant adenoviruses. Results The recombinant adenoviruses containing the interest gene and being able to infect ECV 304 cells were obtained. Overexpression of wild type SPK gene in ECV 304 cells increased the endogenous SPK activity, whereas overexpression of mutant SPK (SPK DN ) inhibited intracellular SPK activity. Conclusion Recombinant adenoviral vectors can mediate interesting gene expression in cells.
