应用分子生物学方法将人β_内啡肽融合基因序列克隆入腺病毒穿梭质粒pDC312_AAVEE,后者与骨架质粒共转染HEK293细胞,包装得到含转基因腺病毒腺相关杂合病毒AdAAV_EE。用PCR方法对杂合病毒进行鉴定,TCID50法测定病毒滴度。杂合病毒感染体外培养细胞观察转基因表达,ELISA法测定培养液中表达产物浓度。PCR法证实转基因正确插入了杂合病毒基因组内,且没有野生型病毒污染,病毒滴度为1.29×1010PFUmL。结果表明转基因从第1天到第14天的表达水平呈上升趋势,第14天达到3141pgmL。 Human β-endorphin fusion gene sequence was cloned into adenovirus shuttle plasmid pDC312_AAVEE by molecular biology method. The latter was co-transfected with backbone plasmid into HEK293 cells and packaged to obtain AdAAV_EE containing adeno-associated virus. Heterozygous viruses were identified by PCR method and TCID50 method was used to determine the virus titer. Heterozygous virus was used to infect cells in vitro to observe the transgene expression. The concentration of the expressed product in the culture medium was determined by ELISA. PCR confirmed that the transgene was correctly inserted into the genome of the hybrid virus, and no wild-type virus was contaminated. The virus titer was 1.29 × 1010 PFU mL. The results showed that the expression level of the transgene increased from day 1 to day 14, reaching 3141 pgmL on the 14th day.