【作者】 陆叶； 张孝林； 郑小坚； 薛仁宇； 曹广力； 贡成良；

【Author】 LuYe1 Zhang Xiao-lin2,Zheng Xiaojian2 Xue Ren-yu2,Cao Guang-li2,Gong Cheng-liang2 (1School of Pharmacy,2.School of Basic Medica l and Bioscience,Soochow University,Jiangsu Suzhou 215123,China)

【机构】 苏州大学药学院； 苏州大学基础医学与生物科学学院；

【摘要】 目的探讨IL-28A基因通过Bac-to-Bac家蚕杆状病毒表达系统在家蚕BmN细胞及蛹中进行表达。方法应用PCR、基因克隆及共转染等技术将IL-28A插入到家蚕杆状病毒多角体启动子的下游,构建重组病毒;并在家蚕细胞及家蚕蛹中表达IL-28A。用ELISA试剂盒检测感染24h、48h、72h、96h后家蚕培养细胞及蛹中(24h、48h、72h、96h、120h、144h)IL-28A蛋白的表达情况。结果获得含有IL-28A基因片段的重组杆状病毒表达载体pFastBacTMDual-IL28A能在家蚕细胞中表达IL-28A蛋白,感染后的培养细胞中可见绿色荧光;感染pFastBacTMDual-IL28A的家蚕蛹血淋巴能高效表达IL-28A蛋白。ELISA表明感染重组病毒96h的BmN细胞蛋白表达量最高为11.4μg/106细胞,蛹的血淋巴中120h最高为33.2μg/ml,感染重组病毒120h蛹粉为8.488mg/g。结论 IL-28A基因在家蚕蛹中能获得高效表达,为进一步研制IL-28A的口服制剂奠定科学的理论基础。更多还原

【Abstract】 Objective To express interleukin-28A gene in Silkworm/Baculovirus expression vector system(Bac-to-Bac).Methods Interleukin-28A gene was amplified by polymerase chain reaction (PCR) and then inserted into the downstream of polyhedron promoter of the BmNPV.Recombinant virus was constructed by gene cloning,costransfection,and plaque purification.Then the interleukin-28A was expressed in BmN cells and silkworm pupae.The expression of interleukin-28A in BmN cell (harvested at 24 h post infection) was detected by direct green fluorescence assay,and the expression of interleukin-28A in silkworm pupa (harvested at 96 h post infection) was detected by direct green fluorescence assay.Results The recombinant baculovirus expression vector Bacmid-EGFP-IL28A containing interleukin-28A was constructed successfully.The interleukin-28A protein was expressed in BmN cells and detected as green fluorescence assay.The interleukin-28A protein was efficiently expressed in silkworm pupae infected with the recombinant virus Bacmid-EGFP-IL28A.ELISA results showed that the amount of expressed protein reached 11.4μg/106 cells in BmN cells at 96h post infection and 33.2μg/ml in silkworm pupae larvae at 120h post infection and 8.488mg/g in silkworm pupae powder at 120h post infection.Conclusion The interleukin-28A could be expressed at higher level in silkworm pupae with Silkworm/Bac-to-Bac expression vector system,and provide scientific theoretic basic and might be used as a candidate for studying oral vaccine of interleukin-28A.更多还原