绵羊肺腺瘤病毒囊膜蛋白单克隆抗体的制备及双抗体夹心ELISA方法的初步建立  

Preparation of the Monoclonal Antibody against Envelope Protein of JSRV and Development of Double Antibody Sandwich ELISA for JSRV

作　　者：刘月[1] 张宇飞[1] 孙晓林[1] 刘淑英[1,2] 

机构地区：[1]内蒙古农业大学兽医学院,呼和浩特010018 [2]农业部动物临床诊疗技术重点实验室,呼和浩特010018

出　　处：《畜牧兽医学报》2015年第5期800-807,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31160493;31360597);内蒙古科技创新引导基金项目(20130224);教育部博士点基金博导类项目(20111515110008)

摘　　要：为制备抗绵羊肺腺瘤病毒(JSRV)囊膜蛋白(ENV)胞质尾区(CT)的单克隆抗体,利用生物信息学分析JSRV囊膜蛋白氨基酸序列,根据分析结果合成多肽,将此多肽分别连接钥孔血蓝蛋白(CT-KLH)和牛血清白蛋白(CT-BSA),用CT-KLH作为抗原免疫BALB/c小鼠,取免疫小鼠的脾细胞与骨髓瘤细胞融合。建立间接ELISA方法筛选分泌抗CT蛋白单克隆抗体的杂交瘤细胞,获得1株能稳定分泌抗CT蛋白单克隆抗体的杂交瘤细胞株,命名为8E5,亚类为IgG2a/κ。间接ELISA检测培养上清效价为6.4×103,腹水效价为1×105。Western blot及免疫组化结果显示,这株杂交瘤细胞分泌的单克隆抗体能识别JSRV抗原,与其发生特异性反应。试验结果证明,单抗8E5是绵羊肺腺瘤的重要诊断试剂。应用8E5细胞株分泌的单克隆抗体建立JSRV-ENV的双抗体夹心ELISA检测方法,该ELISA检测方法特异性、重复性和敏感性良好,为快速检测诊断绵羊肺腺瘤和研究囊膜蛋白功能奠定了基础。In order to prepare monoclonal antibody against the cytoplasmic tail of the envelope protein of Jaagsiekte sheep retrovirus(JSRV),the amino acid sequence of the JSRV envelope protein was analyzed by bioinformatics to synthetize polypeptide.Then the polypeptide was connected with Keyhole Limpet hemocyanin(CT-KLH)or bovine serum albumin(CT-BSA),respectively.Polypeptide CT-KLH was immunized to the BALB/c mice as antigen,and the spleen cells of immunized mice were fused with SP2/0myeloma cells.An indirect ELISA was developed to screen positive antibody producing cells using the CT protein.A hybridoma stably secreting MAb designated as 8E5 was obtained against CT protein.The monoclonal antibody belonged to IgG2a/κ.The titers of supernatant and ascites were 6.4×103 and 1×105 as detected by ELISA,respectively.The results of western blot and immunohistochemistry showed that this hybridoma secreted MAb could react specifically with the JSRV.The results suggested that the MAb-8E5 is important diagnostic reagents for detection of ovine pulmonary adenocarcinoma(OPA).A sandwich ELISAwas established by using 8E5 secreted monoclonal antibody.The results indicated that the ELISA possessed good specificity and repeatability,and higher sensitivity.The monoclonal antibody could be used in diagnosis for OPA and further research of envelope protein function.

关 键 词：绵羊肺腺瘤病毒 囊膜蛋白 B细胞表位 单克隆抗体 ELISA 

分 类 号：S852.65[农业科学—基础兽医学]