抗传染性法氏囊病毒VP2蛋白单克隆抗体的制备及鉴定

Preparation and Characterization of Monoclonal Antibodies Against VP2 Protein of Infectious Bursal Disease Virus

传染性法氏囊病(infectious bursal disease,IBD)是由传染性法氏囊病毒(Infectious bursal bursal virus,IBDV)引起的一种高度传染性疾病,以雏鸡法氏囊萎缩、腿肌出血等为特征性病变,给世界家禽产业造成重大经济损失。目前,由新型变异株引起的IBD在我国呈现流行趋势。为建立基于IBDV VP2蛋白的免疫学诊断技术,本研究以新型变异毒株IBDV-LY21/2为模板,扩增其VP2基因,随后构建重组原核表达质粒pCold-Ⅰ-IBDV-VP2并转化BL21(DE3)大肠杆菌进行诱导表达,通过考马斯亮蓝染色和Western blot检测重组蛋白His-VP2的表达情况,并对其进行纯化和浓度测定。将His-VP2免疫BALB/c小鼠后,采用iELISA检测血清抗体效价,最后制备单克隆抗体并对其鉴定。结果显示,获得的重组蛋白His-VP2分子量约为50 kDa,浓度为8 mg/mL;通过两次亚克隆获得3株杂交瘤细胞株1G10F12、3E3E9、4D12G12,且重链均为IgG1型,轻链均为κ型;其中1G10F12、3E3E9可与重组蛋白Myc-VP2产生Western blot和IFA反应,而4D12G12只能与其产生IFA反应。本研究为IBD诊断方法的建立奠定了基础,同时为深入研究VP2蛋白的功能提供生物学工具。

Infectious bursal disease(IBD)is a highly infectious disease caused by Infectious bursal disease virus(IBDV)and the characteristic lesions included atrophy of bursa of Fabricius and bleeding of leg muscles.At present,the rapid spread of a novel IBDV variant has caused great economic losses to poultry industry in China.Therefore,it is significant to develop an effective immunological detection method for rapid diagnosis of IBD.The RT-PCR product of VP2 gene segment was amplified from the RNA extract of the novel variant IBDV-LY21/2 strain.The recombinant prokaryotic expression plasmid pCold-Ⅰ-IBDV-VP2 was transformed into BL21(DE3)cells for protein expression.The expressed recombinant protein was examined by Coomassie blue staining and Western blot.The VP2 protein was expressed in large quantities and purified for preparation of monoclonal antibodies(MAbs).The molecular weight of the recombinant protein His-VP2 was about 50 kDa and its concentration was 8 mg/mL.Then the purified His-VP2 was used to immunize BALB/c mice and the murine serum samples were titrated in indirect ELISA.As a result,Three hybridoma cell lines(1G10F12,3E3E9 and 4D12G12)were obtained after two cycles of subcloning.The heavy chains of these three MAbs were IgG1 and their light chains wereκ.The MAbs 1G10F12 and 3E3E9 reacted with the recombinant protein Myc-VP2 in Western blot and IFA while the 4D12G12 showed reactivity in IFA.This study laid a foundation for development of diagnostic methods for IBD and provided a good tool for further study of the function of VP2 protein.