东北虎免疫球蛋白单克隆抗体的制备与鉴定

Identify and production of monoclonal antibody of siberian tiger’s immunoglobulin

摘要:

摘要：【目的】提纯东北虎免疫球蛋白并制备其单克隆抗体（McAb），为东北虎传染性疾病诊断试剂盒研制、疫苗免疫效果评估及基础免疫学研究奠定基础。【方法】以饱和硫酸铵和重组的Protein G相结合，提纯东北虎免疫球蛋白作为抗原，免疫C57BL/6小鼠，和骨髓瘤细胞系Sp2/0-Ag14相融合制备杂交瘤细胞，ELISA和蛋白免疫印迹筛选及鉴定阳性克隆，应用杂交瘤产生的McAb鉴定提纯免疫球蛋白及制备的McAb免疫学活性，非竞争性ELISA法测定了其亲和常数。【结果】得到三株稳定分泌抗东北虎免疫球蛋白McAb的杂交瘤细胞，产生的抗体全部识别免疫球蛋白的重链，通过对猫泛白细胞减少症病毒株(FPV-HLJ)灭活疫苗免疫东北虎的抗体消长的检测，证明提纯的免疫球蛋白及其McAb的免疫活性。【结论】Protein G 能够用于东北虎免疫球蛋白的提纯，ATD11杂交瘤产生的McAb与抗原有较强的结合能力和免疫学活性，为今后开展东北虎传染病的诊断方法及疫苗研究奠定基础。

Abstract:

Abstract:[Objective] Purify Immunoglobulin (Ig) of Siberian Tiger and prepare monoclonal antibody (mAb) against the Ig, they can be used to develop immunological diagnostic kits for diagnosing infectious disease in Siberian Tiger, and also can be used in basic immunity research. [Methods] The Ig of Siberian tigers was purified with saturated ammonium sulfate combined with recombinant Protein G. The C57BL/6 mice were immunized with the purified Ig. Spleno-cytes of the mice immunized were collected and fused with the mouse myeloma cell line (Sp2/0-Ag14). The positive hybridoma clones were selected by ELISA and were identified by western blot. The sandwich ELISA was used to detect immunocompetence of the purified Ig and the mAb. [Results] We obtained three mouse hybridoma clones in this study can produce mAbs against Ig of Siberian Tiger. The derived McAbs can recognize Ig heavy chain of Siberian Tiger specifically. The biological activity of the Ig and obtained McAbs also can be identified by detecting the antibody induced by panleukopenia virus (FPV-HLJ) vaccine in Siberian Tiger. The antibody also would be useful for assess the vaccine effecacy against the infectious disease on the Siberian Tiger. [Conclusion] Protein G can be used in Ig purification of Siberian Tiger. The obtained McAbs from the hybridoma ADT11 in this study owned strong ability to bind Ig of Siberian Tiger and have a stable immunocompetence. They can be used to develop diagnostic methods for detecting infectious disease in Siberian Tiger and vaccine research.