鲑鱼降钙素重组杆状病毒载体的构建及表达

Construction and expression of eukaryotic baculovirus expression vector for sCT-gene

目的 利用昆虫细胞杆状病毒系统表达重组鲑鱼降钙素 (recombinantsalmonCalcitonin ,rsCT) ,探索一条真核生物表达生物活性rsCT的新途径。方法 将鲑鱼降钙素基因重组到 pFastBac杆状病毒穿梭载体(baculovirusshuttlevector,Bacmid)中 ,构建重组鲑鱼降钙素杆状病毒表达载体即重组鲑鱼降钙素Bacmid ,后将其转染昆虫细胞Sf9,表达目的蛋白 ,并对表达产物进行分子量及免疫反应性鉴定。结果 证实目的基因在昆虫细胞中获得了表达。

Objective To construct the recombinant bacmid DNA of salmon calcitonin gene and express sCT by transfecting the insect cell line Sf9.Methods The encoding fragment of salmon calcitonin gene was amplified from plasmid pGex-3x-sCT by means of PCR and inserted into baculovirus transfer vector pFastBac1,formed a recombinant plasmid pFastBac1-sCT.And then, pFastBac1-sCT was transposited with baculovirus shuttle vector (bacmid) in the Max Efficiency DH10Bac Competent cells, constructed the expression vector,rsCT bacmid.The rsCT bacmid was identified by PCR and used to transfect the Sf9 insect cells to produce the interesting peptide.Results The expression product was identified and estimated by Tricine-SDS-PAGE and radioimmunological assay.Conclusion The sCT gene was successfully expressed in insect cells by the baculovirus eukaryotic expression vector system (BEVS).