RHDV VLPs单克隆抗体的制备及其识别表位的初步定位

doi:10.7668/hbnxb.2015.05.011

华北农学报 ›› 2015, Vol. 30 ›› Issue (5): 65-70. doi: 10.7668/hbnxb.2015.05.011

RHDV VLPs单克隆抗体的制备及其识别表位的初步定位

宋艳华, 胡波, 范志宇, 彭伟, 魏后军, 薛家宾, 徐为中, 王芳

江苏省农业科学院兽医研究所, 农业部兽用生物制品工程技术重点实验室, 国家兽用生物制品工程技术研究中心, 江苏南京 210014

收稿日期:2015-07-20 出版日期:2015-10-28
通讯作者: 王芳(1972-),女,新疆伊宁人,研究员,博士,主要从事畜禽传染病免疫机理及防控技术研究。
作者简介:宋艳华(1985-),女,山东聊城人,助理研究员,博士,主要从事畜禽传染病防控研究。
基金资助:
江苏省自然科学基金项目(BK20140740);江苏省农业科技自主创新资金项目(CX(13)5029);现代农业产业技术体系建设专项资金项目(CARS-44)

Preparation and Primary Mapping the Epitopes of Monoclonal Antibodies Against RHDV VLPs

SONG Yan-hua, HU Bo, FAN Zhi-yu, PENG Wei, WEI Hou-jun, XUE Jia-bin, XU Wei-zhong, WANG Fang

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Research Center of Veterinary Biologicals Engineering and Technology, Nanjing 210014, China

Received:2015-07-20 Published:2015-10-28

摘要/Abstract

摘要： 为了筛选兔出血症病毒(RHDV)衣壳蛋白(VP60)的特异性单抗,并进一步分析单抗识别表位的分布情况,将重组杆状病毒rAcV-Bac-VP60 接种Sf9昆虫细胞,收获细胞培养物,电镜观察显示重组VP60蛋白获得有效表达,并可组装成病毒样粒子(VLPs)。将RHDV VLPs作为免疫原与等量弗氏佐剂乳化,免疫BALB/c小鼠,取脾细胞与SP2/0 骨髓瘤细胞融合,经3次亚克隆后,获得11株能稳定分泌抗RHDV VLPs抗体的阳性杂交瘤细胞株。间接ELISA、IFA和Western Blot鉴定结果表明,这11株单克隆抗体均能够特异地识别RHDV VLPs和天然RHDV。11株单抗均为IgG1,其中1D4和3F7的轻链为Lambda型,其余均为Kappa型。截短表达结果显示,单抗5A3针对RHDV VP60的NTA区,5F3针对RHDV VP60的S区,单抗1B8、1D4、3D11、3F7、4C2、4G2、5G2、5H3和6B2针对RHDV VP60的P区。研究为RHDV VLPs抗原表位的鉴定和RHDV结构功能的研究奠定了基础,同时为RHDV的检测和新型疫苗研究提供物质基础。

关键词: RHDV VLPs, 单克隆抗体, 表位, 初步定位

Abstract: To select specific mAbs for the capsid protein(VP60)of Rabbit hemorrhagic disease virus(RHDV)and further analysis the distribution of antigenic epitopes recognized by mAbs,the recombinant baculoviruses rAcV-Bac-VP60 infected Sf9 insect cell monolayers to effectively express the recombinant VP60 protein,which could self-assemble into VLPs.To prepare monoclonal antibody(MAbs)specific to RHDV VLPs,BALB/c mice were immunized with the recombinant VP60 protein emulsified with Freund's adjuvant.Splenocytes from immunized mice were fused with SP2/0 murine myeloma cells on day 3 after the last injection.After three times subcloning,eleven monoclonal antibodies were screened,which could recognize RHDV VLPs and native RHDV.The subtypes of 1D4 and 3F7 belonged to IgG1-λ.And other MAbs belonged to IgG1-κ.Western Blot results indicated that 5A3 recognized the NTA domain of VP60 protein,5F3 recognized the S domain of VP60 protein and 9 MAbs(1B8,1D4,3D11,3F7,4C2,4G2,5G2,5H3 and 6B2)recognized the P domain of VP60 protein.These MAbs will be useful for mapping the epitopes of RHDV VLPs and the research of the structure and function of RHDV in the future,which will also contribute to the detection of RHDV and its novel vaccine development.

Key words: RHDV VLPs, MAbs, Epitope, Primary mapping

中图分类号:

S858.2912.65

宋艳华, 胡波, 范志宇, 彭伟, 魏后军, 薛家宾, 徐为中, 王芳. RHDV VLPs单克隆抗体的制备及其识别表位的初步定位[J]. 华北农学报, 2015, 30(5): 65-70. doi: 10.7668/hbnxb.2015.05.011.

SONG Yan-hua, HU Bo, FAN Zhi-yu, PENG Wei, WEI Hou-jun, XUE Jia-bin, XU Wei-zhong, WANG Fang. Preparation and Primary Mapping the Epitopes of Monoclonal Antibodies Against RHDV VLPs[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(5): 65-70. doi: 10.7668/hbnxb.2015.05.011.

http://www.hbnxb.net/CN/Y2015/V30/I5/65

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