Construction and identification of the incompetent-replication adenovirus carrying the fusion gene composed of prepropeptide of mouse nerve growth factor and human beta-endorphin gene / 中华麻醉学杂志

ABSTRACT

Objective To construct and identify the incompetent-replication adenovirus carrying the fusiongene composed of the encoding gene of prepropeptide of mouse nerve growth factor(PN)and human beta-endorphin(?-EP)geue.Methods The gene segments of PN obtained from total RNA of the submandibular glandof a 2-week old Kumning mouse were amplified by RT-PCR and joined with the segment of ?-EP to form the fusiongene which was sequenced.The fusion gene contained in the incompetent-replication adenovirus was formed in theBJ-Ad Easy-1 susceptible cells and identified by PCR so as to choose the positive clone without wild vectors.Thecorrect clone was amplified and purified.The titers of adenovirus were determined using the specific 50% tissueculture infection dosage(TCID 50)method.Three days after the adenovirns was transferred into the cultured A431cells,RT-PCR was performed to showed the transcribed mRNA of this fusion gene and the intracellular ?-EPexpression was quanlitatively detected by inummo-histological method.Finally the concentration of human ?-EP inthe culture medium was determined by quantitative radio-immunoassay on 1st,3rd and 7th day afterinfeetion.Results The sequence of the fusion gene was correct.The titer of recombinant adenovirus Ad-NEP was1.5?10~10 pfu/ml.Three days after infection a 475 bp segment was amplified by RT-PCR and abundant orangegranules were shown in the infected cell.The ?-EP concentration in the culture medium was significantly higher inAd-NEP group than in the control group on 1st,3rd and 7th day(P