抗山羊IgMμ链单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibodies against hircine IgMμ chain

将构建的山羊IgMμ链的原核表达重组质粒pET30a-IgMμ转化至大肠杆菌BL21中,诱导表达获得重组蛋白His-IgMμ;以Ni-NTA亲和层析法纯化His-IgMμ重组蛋白为抗原免疫BALB/c小鼠,经细胞融合、抗体筛选和细胞克隆等杂交瘤细胞技术,筛选能稳定分泌抗山羊IgMμ链单克隆抗体的杂交瘤细胞株;采用小鼠腹腔诱生腹水的方法制备出抗IgMμ链单抗,并鉴定单抗的生物学特性。结果,成功构建了重组表达载体pET30a-IgMμ,诱导表达、纯化得到了目的蛋白;获得了4株能稳定分泌抗山羊IgMμ链单克隆抗体的杂交瘤细胞株,4株单抗腹水的效价均达到了1×10^（-4）以上,其中1D8、5D1和6G10株单抗亚型为IgG1,9H8株的亚型为IgG 2a,轻链均为κ;1D8、5D1、6G10和9H8的亲和力平衡解离常数KD分别为1.87×10^（-11）、8.46×10^（-7）、4.26×10^（-9）和4.07×10^（-10）;经Western-blot鉴定,单克隆抗体6G10能特异性识别原核表达的HisIgMμ蛋白。上述研究结果表明,本研究获得了4株高亲和力和效价的抗山羊IgMμ链特异性单克隆抗体,这为山羊感染性疾病的早期诊断、预报预警和防控提供了基础条件。

In this study,a recombinant plasmid pET30 a-IgMμ containing the hircine IgMμ chain will be constructed and then transformed into E.coil BL21.The recombinant protein His-IgMμ will be expressed and purified via Ni-NTA affinity chromatography and then used as an immunogen.Hybridoma lines secreting McAbs specific for hircine IgMμ chain will be obtained via cell fusion,antibody detection and cell cloning.Ascitic fluids containing the anti-chain McAbs will be prepared by induction of hybridomas in mice,and the biological characteristics of McAbs will be identified.In result,we have successfully constructed the recombinant plasmid pET30 a-IgMμ and prepared His-IgMμ protein by expression and purification. Four hybridoma lines producing McAbs specific for hircine IgMμ chain were acquired.The titers of ascitic fluids of McAbs were up to 10-4.McAbs 1 D8,5 D1 and 6 G10 belong to IgG1 isotype,9 H8 belongs to IgG2 a isotype,and all McAbs belong to κ light chain.The equilibrium dissociation constant（KD） of McAbs 1D8,5 D1,6 G10 and 9H8 were 1.87×10^（-11）,8.46×10^（-7）,4.26×10^（-9） and 4.07×10^（-10）,respectively.McAb6G10 could specifically react with His-IgMμ protein by Westernblot.In conclusion,four specific McAbs with high affinity and titer against hircine IgMμ chain were obtained,which provide material for early diagnosis,forecast and early warning,prevention andcontrol of infectious hircine diseases.