[Construction and analysis of in vitro expression of adenovirus/adeno-associated hybrid virus containing the fusion gene of human beta-endorphin].
The fusion gene of human beta-endorphin was cloned into the shuttle plasmid pDC312-AAVEE with the method of molecular bilology. The latter and genomic plasmids were cotransfected into HEK293 to package the Adenovirus/Adeno-associated hybrid virus containing fusion gene of human beta-endorphin. The hybrid virus was identified with the method of PCR. The titer of proliferated virus, after purified, was determined by TCID50. The expression of transgene was studied after the hybrid virus infected the cultured cells, through testing the concentration of expressed product in the culture liquid by ELISA. It was identified that the sequence of fusion gene of human beta-endorphin was correctly inserted into the genome of hybrid virus, and not contaminated by wild type virus. The titer of Ad/AAV-EE is 1.29 x 10(10) PFU/mL after purification. The ascending trend of transgene expression was observed from the 1st to the 14th day, and the protein concentration reached 3141 pg/mL at the 14th day.
Xu
Xue-Wu
XW
Department of Anesthesiology, Eastern Hepatobiliary Hospital, Second Medical Military University, Shanghai 200438, China. xu_xw2002@yahoo.com.cn
Yu
Wei-Feng
WF
Cui
Zhen-Fu
ZF
Wang
Xing-Hua
XH
Li
Quan
Q
Wu
Fei-Xiang
FX
Dong
Gang
G
Jiang
Li-Hua
LH
D000256:Adenoviridae / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D015316:Genetic Therapy; D006801:Humans; D006824:Hybridization, Genetic; D010957:Plasmids; D019076:Transgenes; D001615:beta-Endorphin / Q000235:genetics*
D001615:beta-Endorphin