利用杆状病毒载体在棉铃虫血细胞中表达大肠杆菌β-半乳糖苷酶

High Level Expression of Escherichia coli β-galactosidase in Heliothis armigera Cells by Using a Baculovirus Vector

用携带大肠杆菌β-半乳糖苷酶基因的杆状病毒转移载体pAc360-β-gal与野生型的苜蓿丫纹夜蛾(AcNPV)DNA同时转染草地夜蛾细胞,经X-gal筛选和空斑纯化得到重组型杆状病毒AcNPV-β-gal。该重组病毒能有效地感染棉铃虫血细胞系,并高效表达出受AcNPV多角体蛋白启动子控制的、具有生物活性的外源基因产物——β-半乳糖苷酶。80%以上的酶蛋白能分泌到细胞外,培养液中酶活可达每毫升50000单位以上,约相当于170μg酶蛋白。用SDS-聚丙烯酰胺凝胶电泳分离表达产物和β-半乳糖苷酶在凝胶上的特异性显色反应分析,重组病毒在棉铃虫血细胞中表达的AcNPV多角体蛋白——大肠杆菌β-半乳糖苷酶融合蛋白是以5种活性的多聚体或复合物形式存在的。

The E. colt β-galactosidase gene carried by a baculovirus vector pAc360-β-gal was transferred into the AcNPV genome by cotransfection of S. frugiperda cells. A recombinant baculovirus, AcNPV-β-gal was obtained by means of X-gal selection and plaque purification. This recombinant baculovirus could effectively infect established H. armigera cell line and express the biologically active β-galactosidase under the control of the baculoviral polyhedrin promotor. More than 80% the β-galactosidase was secreted from infected H. armigera cells and over 50 000u/ml of activity, equal to calculate 170μg/ml of protein was detected in infected cells medium. Seperation with SDS-PAGE and detection of β-galactosidase activity directly on gels showed that the products of the foreign gene expressed in H. armigera cells infected with AcNPV-β-gal existed in the forms of five active polymers or complexes. By comparison of abilities of the β-galactosidase expression with wildly used S. frugiperda cells, we think H. armigera cells might be developed as an alternative to S. frugiperda cells and the H. armigera cells——AcNPV might become a new insect cells——baculovirus expression system.