马铃薯Y病毒小鼠中和抗体轻链基因的克隆及序列分析  

Cloning and Sepuencing of Kappa Light Chain Gene of a Mouse Monoclonal Antibody Directed Against Potato Virus Y

作　　者：刘德虎[1] 郭军[1] 陈三风[1] 梁华[1] 李刚强[1] 

机构地区：[1]中国农业科学院生物技术研究中心分子生物学研究室,北京农业大学生物学院微生物学系

出　　处：《农业生物技术学报》1995年第2期86-92,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金;瑞典科学基金

摘　　要：从大量繁殖的Ｄ７３杂交瘤细胞中，提取制备其基因组ｍＲＮＡ，并以此为模版，经反转录途径获得基因组ｃＤＮＡ，随后将其插入到λｇｔｌｌ中，构建了马铃薯Ｙ病毒小鼠单克隆抗体ｃＤＮＡ基因文库。通过免疫原位杂交，从该基因文库中筛选出含有抗体轻链基因的阳性克隆，进一步将其插入到ｐＧＥＭ－７Ｚｆ（＋）质粒中，经酶谱和ＤＮＡ序列分析确定，完整的轻链基因被获得。该基因含有９５６个核苷酸碱基［不包括Ｐｏｌｙ（Ａ）尾巴］，其中包括５’一端非编码区３１个核苷酸碱基，编码轻链信号肽的５７个核苷酸碱基，编码成熟蛋白的６５７个核苷酸碱基和３’一端非编码区的２１１个核苷酸碱基。与已知的其它ｋ轻链（ｋａｐｐａ）基因（ＭＲＫ１６）相比，核苷酸序列同源性为９８．７％，由核苷酸序列所推导出的氨基酸的序列同源性为９７．０％。该同源性在不同功能区域之间存在着差异，其中在保守区（ＣＬ），氨基酸的序列同源性为１００％，而在可变区（ＶＬ），同源性为９３．７％，其变异主要发生在三个不同的区域，即所谓的抗原识别位点（ＣＤＲ）。该蛋白被认为对马铃薯Ｙ病毒是特异性的，因为Ｄ７３杂交瘤细胞只能够表达一种轻链蛋白。A cDNA library was constructed inLgtll vectors,complementary to the mRNA isolatedfrom a mouse hybridoma raised against potato virus Y(PVY).Thirty cDNA clones were selected from thecDNA library by in situ immunohybridization with goat anti-mouse kappa-chain-specific antibody conjugat-ed to alkaline phosphatase from which one clone k6,having the largest insert was characterized by se-quence analysis.The result showed that the immunoglobulin messenger RNA corresponding to k6 was 956nucleotides in length excluding the poly(A)region,among which 31 bases code for the 5’non-codingregion,57 for the leader sequence of the protein,657 for the mature protein and 2ll for the3’non-codingregion. Comparison of deduced amino acid sequences of the protein and other kappa light chains showedthat they share a 100%identity in their constant regions (CL) and 93.7%identity in their variable regions(VL).The kappa light chain encoded by k6 was considered to be specific to PVY since only one type oflight chain was expressed in the hybridoma.

关 键 词：马铃薯Y病毒 单克隆抗体 基因克隆 序列分析 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]