施马伦贝格病毒核衣壳蛋白的表达及单克隆抗体制备

Expression of the Nucleocapsid Protein of Schmallenberg Virus Using the Bac-to-Bac~ Baculovirus Expression System and Preparation of Monoclonal Antibodies against This Protein

为表达与纯化具有天然构象的施马伦贝格病毒(Schmallenberg virus,SBV)核衣壳(N)蛋白,并制备其单抗(McAb),本研究在SBV-N基因的N端加入6个组氨酸(6×His)标签后,将其克隆至杆状病毒表达载体pFastBac^(TM)1中,构建重组供体质粒pFastBac-His-SBV-N。将pFastBac-His-SBV-N转化DH10Bac E.coli,通过蓝白斑筛选获得重组杆粒rBacmid-His-SBV-N。将rBacmid-His-SBV-N转染Sf9昆虫细胞制备表达His-SBV-N融合蛋白的重组杆状病毒,借助Ni-NTA琼脂糖纯化重组杆状病毒感染Sf9细胞中的His-SBV-N蛋白。以纯化的HisSBV-N免疫BALB/c小鼠制备McAb,利用ELISA叠加试验检测McAb抗原识别位点的异同,并采用高碘酸钠法对识别不同抗原表位的McAb进行辣根过氧化物酶(HRP)标记。最终获得了4株识别不同抗原表位的McAb(2A11、2E1、4H11和6E12)并进行了HRP标记;亚型鉴定表明,2A11为IgG1亚型,2E1、4H11和6E12均为IgG2b亚型;间接免疫荧光试验证实,4株McAb均能够识别稳定表达SBV-N蛋白的BHK-21细胞系;Western blot进一步表明,HRP标记的4株McAb均与His-SBV-N蛋白发生特异性反应。His-SBV-N融合蛋白及其McAb的成功制备,为施马伦贝格病血清学检测方法的建立提供了良好的生物材料。

The objective of this study was to express and purify the nucleocapsid（N）protein of Schmallenberg virus（SBV）with the native conformation,and to prepare its monoclonal antibodies（McAbs）.To this end,the SBV-N gene with an inserted coding sequence for an N-terminal hexahistidine（6×His）tag was cloned into the pFastBac^（TM）1 vector.The resulting recombinant donor plasmid,pFastBac-His-SBV-N,was then transformed into DH10 Bac E.coli competent cells.After selection with the blue/white screen,the recombinant bacmid,rBacmid-His-SBV-N,was transfected into Sf9 insect cells to generate recombinant baculoviruses that express the HisSBV-N fusion protein.After amplifying the recombinant baculoviral stocks on Sf9 cells,His-SBVN fusion protein was purified using nickel nitrilotriacetic acid（Ni-NTA）agarose.The purified protein was then used to immunize BALB/c mice to prepare SBV-N-specific McAbs,and an indi-rect double-antibody binding ELISA was applied to detect whether the generated McAbs recognize different antigenic sites.The screened McAbs that recognize different antigenic sites were labeled with horseradish peroxidase（HRP）using the sodium periodate oxdization method.Finally,four McAbs（2A11,2E1,4H11 and 6E12）recognizing different antigenic sites were successfully obtained and labeled with HRP.The results of isotype identification demonstrated that 2A11 belongs to IgG1,and 2E1,4H11 and 6E12belong to IgG2 b.Indirect immunofluorescence assays revealed that all of the four McAbs reacted with the BHK-21 cell line stably expressing the SBV-N protein（BHK-21-EGFP-SBV-N）.Western blot analyses further showed that the four HRP-conjugated McAbs reacted positively with the His-SBV-N fusion protein.Taken together,the successful preparation of His-SBV-N fusion protein and its McAbs provide valuable biological materials that can be used in the serological diagnosis of Schmallenberg disease.