慢性蜜蜂麻痹病毒SP1蛋白的原核表达及免疫原性分析  

Prokaryotic Expression and Immunogenicity Analyses of the SP1 Protein of Chronic Bee Paralysis Virus

作　　者：李明[1,2] 孙莉 马鸣潇[2] 侯振中 LI Ming;SUN Li;MA Mingxiao;HOU Zhenzhong(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;College of Animal Husbandry and Veterinary,Jinzhou Medical University,Jinzhou 121001,China)

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030 [2]锦州医科大学畜牧兽医学院,锦州121001

出　　处：《病毒学报》2019年第3期473-478,共6页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:31772760);题目:中华蜜蜂囊状幼虫病毒编码siRNA及其RNAi抑制因子在免疫保护中作用;辽宁省自然科学基金(项目号:20170540375);题目:慢性蜜蜂麻痹病毒结构蛋白免疫原性比较及其优势抗原表位筛选~~

摘　　要：慢性蜜蜂麻痹病毒(Chronic bee paralysis virus,CBPV)主要侵袭成年工蜂,造成患病蜜蜂神经麻痹,丧失飞行能力,出现爬行现象。CBPV据推测有两个结构蛋白SP1和SP2,通过对SP1蛋白的免疫原性探索,为SP1蛋白的功能研究和CBPV生物制剂研制奠定基础。本研究根据大肠杆菌密码子的偏嗜性,对CBPVSP1基因进行优化和合成,将其克隆至原核表达载体pET-32a,获得重组表达质粒pET-Opti-SP1。将重组质粒转化至宿主菌BL21(DE3)中诱导表达,用SDS-PAGE和Western Blot方法对该蛋白进行分析和鉴定,然后对最佳表达条件进行摸索。在此基础上,用纯化后的SP1蛋白免疫接种BALB/c小鼠,同时设置纯化病毒组、pET-32a对照组和空白对照组,检测免疫小鼠的淋巴细胞增殖指数(SI)和血清中抗体水平。结果表明,首次成功表达和纯化了重组SP1蛋白,且该蛋白能诱导产生特异性抗CBPV的抗体,促进淋巴细胞的增殖和提高IL-4、IL-10、IFN-γ水平,具有较好的免疫原性。本研究为进一步研究CBPV SP1蛋白的功能和防治CBPV生物制剂的研发提供了帮助。Chronic bee paralysis virus(CBPV)mainly invades adult worker bees,causing nerves paralyzed by sick bees,losing flight ability and crawling.It is speculated that there are two structural proteins SP1 and SP2 in CBPV.The immunogenicity of the SP1 protein was explored.We wished to lay a foundation for the functional study of the SP1 protein and development of biologic agents against the CBPV.The SP1 gene of the CBPV was optimized and synthesized according to a preference for the Escherichia coli codon.The SP1 gene was cloned into the pET-32 a vector,and the recombinant prokaryotic expression plasmid pET-Opti-SP1 was obtained.The recombined plasmids were transformed into BL21(DE3)to induce expression,respectively.Proteins were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western Blot,and then the optimal expression conditions were explored.On this basis,BALB/c mice were immunized with purified SP1 protein.A"pure virus"group,pET-32 a group,and a blank control group were created.Then,the antibody level,lymphocyte proliferation index,as well as levels of interleukin(IL)-4,IL-10 and interferon(IFN)-γin the immunized mice were determined.We first showed that the recombinant SP1 protein was expressed and purified.The recombinant SP1 protein could induce production of specific antibodies against it,promote lymphocyte proliferation,increase levels of IL-4,IL-10,and IFN-γ,and have high immunogenicity.The present study provided insights for functional studies of the SP1 protein of the CBPV,as well as development of biologic agents for the prevention and treatment of CBPV infection.

关 键 词：慢性蜜蜂麻痹病毒(CBPV) 慢性蜜蜂麻痹病(CBPD) 结构蛋白 原核表达 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学] S895[农业科学—兽医学]