基于定量PCR技术的AcNPV-CarE病毒液滴度测定及酯酶表达条件优化

Determination of the Titres of AcNPV-CarE with Quantitative PCR and Optimization of the Conditions for Esterase Expression in vitro

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 produce native carboxylesterases（CarEs） protein in vitro in cotton bollworm（Helicoverpa armigera） and to understand its metobolic detoxification of some chemical insecticides such as OPs,synthetic pyrethroids（SPs） and carbamates（CBs）,the CarE001H of H.armigera was expressed in Sf9 cells with baculovirus vector from Autographa californica nucleopolyhedrosis virus（AcNPV）.The titres of the recombinant virus（AcNPV-CarE） were determined,using quantitative real time PCR（Q-PCR）.The expression of CarE001H was optimized based on the result of native PAGE stained with α-naphthyl acetate and the change trend of total cell density with a different multiplicity of infection（MOI） and a time course for cell harvesting.The results showed that the P3 and P4 stocks had high titres（2×108 pfu/mL）,and thus could be used to infect Sf9 cells with an MOI of 2 and a cell density of 3×106 cell/mL to produce the maximum yields of native CarE protein when the cells were harvested after two days＇ infection.The above results should be helpful for the heterologous and eukaryotic expression of CarE genes and the preparation of active CarEs protein in the future.