猫CD3单克隆抗体的筛选与鉴定

Screen and identification of monoclonal antibodies against feline CD3

为研究猫CD3的生物学功能、猫淋巴瘤和自身免疫性疾病的诊断及治疗方法,通过克隆构建含猫CD3胞外区基因的重组质粒,将ExpiCHO-S细胞和Expi293细胞表达的猫CD3蛋白分别作为免疫原和筛选抗原,利用杂交瘤技术制备抗猫CD3单克隆抗体(m Ab),对其抗体亚型和杂交瘤细胞株稳定性进行鉴定和分析,IFA、Western-blot、免疫组化(IHC)和流式细胞术(FCM)分析其生物学特性。结果显示,成功制备了2株可持续稳定分泌抗猫CD3 m Ab杂交瘤细胞株,命名为2C9和3E1;亚型鉴定结果显示均为Ig G1型,轻链类型均为κappa型。IFA和Western-blot结果显示,2株抗猫CD3 m Ab均可与体外表达的猫CD3蛋白发生特异性结合。IHC和FCM结果显示只有2C9 m Ab可特异性识别猫脾、肠道淋巴组织及猫外周血单核淋巴细胞(PBMCs)中的T淋巴细胞。结果表明,本研究制备的猫CD3 m Ab为后续猫CD3分子的功能研究、猫淋巴瘤和自身免疫性疾病的诊断及治疗方法的建立奠定了基础。

In order to study the biological function of feline CD3,diagnostic and therapeutic methods of lymphoma and autoimmune diseases in feline,recombinant plasmid containing feline CD3 extracellular domain gene was cloned and constructed,feline CD3 protein expressed by Expi CHO-S cells and Expi293 cells was used as immunogen and screening antigen respectively.The monoclonal antibody against feline CD3 was prepared by hybridoma technique,the antibody subtype was identified and the stability of hybridoma cell line was analyzed.IFA,Western-blot,IHC and FCM were used to analyze its biological characteristics.The study successfully prepared two hybridoma cell lines which can secrete anti-feline CD3 m Ab continuously and stably,named 2C9 and 3E1.The subtype identification results showed that they were all Ig G1 type,and the light chain types were κappa type.IFA and Western-blot results showed that two anti-feline CD3 m Ab could specifically bind to feline CD3 protein expressed in vitro.The results of IHC and FCM showed that only 2C9 m Ab could specifically recognize T lymphocytes in feline spleen,intestinal lymphatic tissue and PBMCs.In summary,the feline CD3 m Ab prepared in this study laid a foundation for the follow-up study on the function of feline CD3 molecule,the diagnosis and treatment of feline lymphoma and autoimmune diseases.