Study On Cloning Of Elastase Gene From Pseudomonas SP-6 And Expression In Insect Cell

Elastase is one of the extended-spectrum endopeptidases with the property of hydrolyzing insoluble elastin,and extendly exists in many tissues and body fluid of mammalians and human. Not only the elastin can be hydrolyzed by elastase,but also do many other proteins such as glutin, HB, albumin, plasma fibrinogen et al.So it was of great economy value and abroad applied in medical treatment,food processing, chemical combination and even environment fathering.At present,domestic resources of elastase is extracted from pork pancreas and was limited by the shock of quantities,therefor the demand exceeds supply for a long time.Foreign research in this aspect has started in early, and has established the prelimimary industrial system of elastase preparing by microbe fermentation.But the elastase produced by microbe fermentation can't give satisfying results in aspects of bio-security and toxicity.So,the resource of elastase as medicine for therapy remained on extracting animal pancreas.In regard to this situation,our research utilized the gene engineering technics to find a novel method for elastese prepareing,and cloned the elastese gene into the vectors to express elastese in insect cell sf21 by baculovirus expression vector system.The reason of using this system is for the bio-security, high productivity and ability of retain the protein activity in nature,because protein produced by baculovirus expression vector system have gotten the confirms of many authorities including FDA.In this research,a 1.5kb gene of elastase was amplified from the genome of a elastase priducing strain Pesudomonas SP-6,and the homology of the PCR product was 100% to the elastase gene of Pesudomonas aeruginosa PAO I repoted in GeneBank.After T-A clone of the PCR product,we got the plasmid pMD18-Ela.To abtain the interim vector pET28a-EIa,the elastase gene was then cut off and transfered into the MCS of vector plasmid pET28-a down stream the 6×His tag.To abtain the baculovirus transfer vector pBacPAK8-His-Ela,the elastase gene and the 6×His tag were cloned into plasmid pBacPAK8. Meanwhile,a positive control of plasmid pBacPAK8-PGF was constructed by cloned the GPF gene into plasmid pBacPAK8 in the same way.The double digestion of BamH I /EcoR I and single digestion of Hind III indicated that the elastase gene and 6×His tag were correctly inserted down stream of the baculovirus polyhedrin promoter.By the medium of lipofectin,the two vector DNAs were transfected into the sf21 cells,along with lined viral DNA.72 hours later,green fluorescent cells in the positive control set was observed and cells in the sample set also got pathological changes.To abtain the recombinant virus BacPAK-Ela and BacPAK-GFP,recovered the cotransfection medium and picked up viral plaques.Tests of cells post infection 96 hours with recombinant virus were made as...