抗菌肽M50基因在Bac-To-Bac杆状病毒系统中的表达

EXPRESSION OF ANTIBIOTIC PEPTIDE OF GENE M50 IN BAC-TO-BAC BACULOVIRUS SYSTEM

将镰形扇头蜱体内的抗菌肽M50全长基因,克隆于杆状病毒转移载体pFastBacHTA中,构建重组质粒pFastBacHTA-M50,并将其转化DH10Bac细胞后,得到重组穿梭质粒reBacmid-M50,再转染到sf9昆虫细胞,进行M50重组蛋白的表达,用Western blot和间接免疫荧光等方法对表达的蛋白进行鉴定。结果表明M50基因在Bac-To-Bac杆状病毒系统中成功表达。带His标签的重组蛋白分子量约为15 kDa,在昆虫细胞中表达的M50蛋白能被抗原核表达的M50蛋白的血清识别,也能被标签His单抗识别。

The M50 Gene encoding antibiotic peptide of tick Rhipicephalus haemaphysaloide was cloned into the Baculovirus transfer vector pFastBacHTA.The recombinant plasmid pFastBacHTA-M50 was transformed into competent cells of E.coli DH10Bac.The purified recombinant shuttle plasmid was transfected into sf9 insect cells to express recombinant protein M50.The recombinant protein expressed was detected by Western blot and immunofluorescent assay.The results showed that M50 protein was successfully expressed in the Bac-To-Bac Baculovirus system.The fusion M50 protein with His tag（His-M50） had a molecular weight of 15kDa.His-M50 was recognized by anti-His monoclonal antibody by Western blot and was positive stained with mouse anti-serum against M50 protein.