用Bac-to-Bac杆状病毒系统表达人生长激素

Expression of Human Growth Hormone Gene in Bac-to-Bac Baculovirus System

利用Bac to Bac杆状病毒载体表达系统将人生长激素 (humangrowthhormone ,hGH)基因cDNA克隆至转移载体pFastBac1中 ,得到pFastBac hGH ,再将其转化进入含穿梭载体Bacmid的受体菌DH10Bac中 ,发生转座作用 ,得到含hGH基因的重组穿梭载体rBacmid hGH .纯化DNA ,直接转染培养的昆虫细胞Sf9,得到重组病毒rAcV Bac hGH .经酶切PCR及Southern杂交鉴定 ,hGH基因正确地插入病毒基因组的多角体蛋白基因启动子下 ,SDS PAGE测得产物蛋白分子量为 2 2kD左右 .用免疫化学发光法测得转染上清中hGH表达水平可达 18μg ml ,与用传统的BEVS表达hGH相比 ,转染上清中hGH表达水平提高 4 0

The Bac to Bac baculovirus expression system is a novel gene expression system that allows the rapid and efficient generation of recombinant baculovirus DNA by site specific transposition in E.coli , rather than homologous recombination in insect cells. The cDNA encoding hGH(human growth hormone) gene was cloned into a transfer vector pFastBac1, and recombinant vector pFastBac hGH DNA was extracted. After transformation, it was introduced into E.coli DH10Bac which included a shuttle vector, Bacmid. By site specific transposition, hGH gene was integrated into Bacmid, and a recombinant shuttle vector was constructed, named rBacmid hGH. The cultured Sf9 cells were directly transfected with the rBacmid hGH DNA, and the pure recombinant baculovirus was obtained, named rAcV Bac hGH. RE assay, PCR and Southern blot analysis revealed that hGH cDNA was correctly inserted into baculovirus genome under the control of polyhedrin promoter. The hGH expression product had a Mr of 22 kD. Immuno Chemiluminescent was used for detecting hGH expression level. The hGH expression level in transfection supernatant was 18 μg/ml, 400 times more than that in the co transfection supernatant by the traditional BEVS(40 ng/ml). Advantages of using this system in the expression control of hGH gene in vitro were also discussed.