铜绿假单胞菌弹性蛋白酶基因的克隆及在昆虫细胞中的表达

Cloning of Elastase Gene From Pseudomonas SP-6 and Expression in Insect Cells

以产弹性蛋白酶的铜绿假单胞菌基因组DNA为模板,经PCR扩增得到约1.5 kb的弹性蛋白酶基因,克隆到质粒pET28 a,连同其上游的6×H is序列转移到pBacPAK8构建成pBacPAK8-H is-E la转移载体。采用L ipofec-tin法,将载体pBacPAK8-H is-E la和亲本病毒BacPAK6共转染Sf21细胞,经空斑纯化分离得到重组病毒BacPAK6-E la。通过双酶切及单酶切鉴定证实弹性蛋白酶基因已插入到病毒基因组的多角体启动子下游,SDS-PAGE检测到在昆虫细胞中表达约34 kD的蛋白条带,以弹性蛋白为底物的生化反应证明了表达产物具有一定的生物学活性。研究结果为弹性蛋白酶的制备提供了新的方法。

A 1.5 kb elastase gene was amplified from Pseudomonas SP-6 genome DNA and inserted into vector pET28a. To obtain the transfer vector pBacPAK8-His-Ela, the fragments of elastase gene and the upstream 6×His sequence were linked into vector pBacPAK8. Sf21 cells were cotransfected with pBacPAK8-His-Ela and BacPAK6 viral DNA mediated by lipofectin, and the recombinant baculovirus BacPAK6-Ela was screened by viral plaques. Identification by double and single enzyme digestion demonstrated that the elastase gene was correctly inserted into baculovirus downstream of polyhedrin promoter. SDS-PAGE analysis showed a band of 34 kD in the expression product of elastase gene in insect cells. A biochemical assay based on elastin for the expression protein verified the biological activities. This result will be helpful to establish a novel system for elastase preparation.