蜜蜂残翼病毒VP2基因密码子优化、原核表达及免疫原性分析

Codon Optimization,Prokaryotic Expression and Immunogenicity of the VP2 Gene of the Deformed Wing Virus

蜜蜂残翼病毒(Deformed wing virus,DWV)是引发世界各地大范围蜂群损失的主要嫌疑对象,间接给农业生产带来了巨大的经济损失。为了探究DWV结构蛋白VP2的免疫原性,深入研究该蛋白的功能和为制备DWV单克隆抗体提供可靠的免疫原,本研究从DWV分离株(GenBank登录号:MF770715)扩增得到VP2基因,同时根据大肠杆菌密码子的偏嗜性对VP2基因进行优化并合成,分别将优化前和优化后的VP2基因克隆到pET-28a载体上,获得重组质粒pET-28a-VP2和pET-28a-opti-VP2,分别转化至宿主菌BL21(DE3)中诱导表达,经SDS-PAGE和Western Blot检测,显示仅优化后的opti-VP2基因高效表达,然后对最佳表达条件进行摸索。在此基础上,对重组蛋白纯化后分3次免疫6周龄的雌性BALB/c小鼠,同时设置纯化病毒组和空白对照组,对免疫小鼠的抗体水平和淋巴细胞增殖指数(SI)以及干扰素(IFN-γ)、白介素IL-2和IL-4水平进行检测。结果表明,重组VP2蛋白能够诱导产生特异性抗体,促进淋巴细胞增殖,提高IFN-γ、IL-2和IL-4水平,具有良好的免疫原性,为深入研究DWV VP2蛋白功能和开发防治DWV的生物制剂提供了帮助。

Deformed wing virus(DWV)is the main suspect causing large -scale honeybee colony losses around the world,which brings huge ecnomic losses to argriculural production indirectly.In order to explore the immunogenicity of the structural protein VP2 of DWV,the function of this protein and provide a reliable immunogen for preparation of a DWV monoclonal antibody.the VP2 gene was amplified from DWV isolate(GenBank accession number:MF770715).The gene was optimized and synthesized according to the preference of E.coli codon,and the VP2 gene was cloned into pET-28a vector.The recombined plasmids pET-28a-VP2 and pET-28a- opti - VP2 were transformed into BL21(DE3)to induce expression,respectively.The expression product was analysed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis(SDS -PAGE)and Western Blot detection.Results showed efficient expression of VP2 after codon optimization.The optimal expression conditions were explored.On this basis,6-week - old female BALB / c mice were immunized three times with purified recombinant protein.A pure -virus group and blank control group were created,and then the antibody level,lym-phocyte proliferation index,as well as levels of interferon(IFN)-γ,interleukin(IL)-2,and IL -4 of immunized mice were determined.The recombinant VP2 protein could induce production of specific antibodies,promote lymphocyte proliferation,increase levels of IFN -γ,IL -2,and IL -4,and had high immunogenicity.These data provide insights into further study on function of the VP2 protein of the DWV,as well as the development of bi- ologic agents for the prevention and treatment of the DWV.