绵羊γ干扰素在家蚕杆状病毒表达系统中的表达及产物的抗病毒活性  

Expression of Sheep Interferon Gamma in Silkworm Baculovirus System and Antiviral Activity Assay of Expressed Product

作　　者：邵丽萍 吕言娜 杨鑫[2] 赵泽 胡小元[2] 李轶女[2] 张志芳[2] 易咏竹[1] 

机构地区：[1]江苏科技大学生物技术学院,江苏镇江212018 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《蚕业科学》2017年第5期809-815,共7页ACTA SERICOLOGICA SINICA

基　　金：国家重点研发计划项目(No.2016YFD0500108);国家自然科学基金项目(No.31670156)

摘　　要：干扰素(IFN)具有广谱的抗病毒作用,利用杆状病毒表达系统安全、高效和后加工过程完整的特点,在家蚕体内表达生产重组绵羊γ干扰素(Ovi IFN-γ)。依据家蚕的密码子偏好性对Ovi IFN-γ基因核苷酸序列进行优化和PCR,并克隆了带有His标签编码序列的Ovi IFN-γ基因序列Ovi IFN-His-Tagged-γ,构建分别插入Ovi IFN-γ和Ovi IFN-His-Tagged-γ基因的重组病毒。将2种重组病毒感染家蚕5龄幼虫,Western blot检测蚕体血淋巴中重组Ovi IFN-γ和Ovi IFN-His-Tagged-γ成功表达。采用细胞病变抑制法测定蚕体血淋巴中重组Ovi IFN-γ的效价为1.6×106U/m L,重组Ovi IFN-His-Tagged-γ的效价为2.8×105U/m L,前者的抗病毒活性强于后者,稀释至2.0×104倍时,能够完全抑制VSV-GFP病毒感染。上述结果为利用家蚕杆状病毒表达系统在蚕体内高效、安全生产具有抗病毒活性的重组绵羊γ干扰素提供了试验依据。Interferon （IFN） has a broad spectrum of antiviral activity. In order to meet the requirement of using interferon in prevention and treatment of livestock virus disease, the feasibility of preparing recombinant sheep interferon （OvilFN- y） by silkworm baculovirus expression system is explored. The OvilFN-γ gene sequence was optimized according to co- don bias of silkworm and amplified through PCR. The OvilFN-γ gene sequence with His tag sequence （OvilFN-His- Tagged-γ） was cloned and used to construct recombinant viruses containing OvilFN-γ and QvilFN-His-Tagged-γ re- spectively. These two recombinant viruses were used to infect silkworm larvae of the 5th instar. Western blotting revealed successful expression of recombinant OvilFN--y and OviIFN-His-Tagged-γ in hemolymph of silkworm. The recombinant OvilFN-γ in silkworm hemolymph had a titer of 1.6×10^6 U/mL and that of recombinant QvilFN-His-Tagged-γ had a titer of was 2.8×10^5 U/mL as detected by cytopathic inhibition method. The former had a stronger antiviral activity than thelatter. When diluted by 2. OxlO4 times, it could completely inhibit VSV-GFP virus infection. The above results provide a good experimental basis for efficient and safe produc- tion of recombinant interferon γwith antiviral activity in silkworm baculovirus expression system.

关 键 词：绵羊γ干扰素 杆状病毒表达系统 家蚕幼虫 重组蛋白 抗病毒活性 

分 类 号：Q78[生物学—分子生物学] S858.26[农业科学—临床兽医学]