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为了制备具有生物学功能的棉铃虫羧酸酯酶(CarEs)体外表达产物,用于研究其对有机磷等化学杀虫剂的代谢解毒作用,该文尝试将棉铃虫羧酸酯酶基因在草地夜蛾细胞系Sf9和苜蓿银纹夜蛾核型多角体病毒(AcNPV)为载体的表达系统中进行表达,采用实时荧光定量PCR技术测定棉铃虫羧酸酯酶基因和病毒载体重组体(AcNPV-CarE)的病毒液滴度,并通过不同转染系数(MOI)和不同收获时间下培养液中细胞体积单位变化趋势,以及表达产物的非变性聚丙烯酰胺凝胶电泳和α-乙酸萘酯染色结果,优化了棉铃虫羧酸酯酶在Sf9细胞中的最佳表达条件.结果表明:重组病毒液P1stock在Sf9细胞中扩大培养至第三代以后的病毒液(P3和P4stock)滴度均在2×108 pfu/mL以上;采用P4stock重组病毒液,在MOI为2,Sf9细胞体积单位为3×106个/mL,转染后48h收获细胞沉淀,可得到具有酶活的最大量羧酸酯酶表达产物.上述结果为棉铃虫羧酸酯酶的异源真核表达和具有天然酶活表达产物的制备提供了有价值的参考.
In order to study the biological detoxification of CarE (Carbosterase Carboxylesterase) (CarE), which is used to study the metabolic detoxification of chemical insecticides such as organophosphorus, The expression of Carboxylesterase gene of Helicoverpa armigera (HNN) and the recombinant adenovirus vector (AcNPV-1) were detected by real-time fluorescence quantitative polymerase chain reaction (PCR) CarE) were used to detect the virus titer. The changes of cell volume units in culture medium with different transfection coefficient (MOI) and different harvesting time, as well as the non-denaturing polyacrylamide gel electrophoresis and α-naphthyl acetate As a result, the optimal conditions for the expression of carboxylesterase in Helicoverpa armigera were optimized.The results showed that the titer of the virus fluid (P3 and P4stock) of the P1 virus in Sf9 cells expanded to the third generation was 2 × 108 pfu / mL. Using P4stock recombinant virus liquid, the cell pellet was harvested at a MOI of 2 and a Sf9 cell volume of 3 × 10 6 cells / mL. After 48 h of transfection, the maximum amount of carboxylate ester Enzyme expression product Helicoverpa armigera heterologous eukaryotic expression of the carboxylesterase activity and natural expression of the product of Preparation provide valuable reference.