溶藻弧菌抗独特型抗体单链抗体在毕赤酵母中的表达  

Immunogenicity of single chain variable fragment of anti-idiotype monoclonal antibody against Vibrio alginolyticus and its expression in Pichia pastoris

作　　者：付建芳[1] 涂艳阳[2] 秦红[3] 黄威权[3] 

机构地区：[1]第四军医大学西京医院内分泌科 [2]解放军63870部队医院 [3]第四军医大学组织胚胎学教研室

出　　处：《免疫学杂志》2009年第5期491-494,共4页Immunological Journal

基　　金：国家高技术研究发展计划(863)资助项目(2006AA100308)

摘　　要：目的构建溶藻弧菌抗独特型抗体的单链抗体(scFv)基因的毕赤酵母分泌型表达载体,并对其表达的蛋白进行免疫原性鉴定。方法从分泌溶藻弧菌抗独特型抗体的杂交瘤细胞株(2F4)中克隆该抗体的重链可变区(VH)基因和轻链可变区(VL)基因(GenBank accession No:DQ011530和DQ011531),并通过基因重组为scFv基因。然后将其克隆入毕赤酵母菌分泌型表达载体pPIC9K中,经序列测定后,电转化入毕赤酵母菌SMD1168中,将经表型筛选和G418抗性筛选的重组酵母菌株进行PCR鉴定。以甲醇进行诱导表达,表达后的重组蛋白利用动物免疫试验进行免疫原性鉴定。结果获得毕赤酵母菌分泌型表达载体pPIC9K-scFv,经G418浓度梯度筛选及PCR鉴定得到高拷贝整合的重组酵母菌株,ELISA测定和动物免疫试验显示抗独特性抗体的scFv具有与溶藻弧菌一样的免疫原性。结论构建溶藻弧菌抗独特型抗体的scFv基因的真核表达载体并成功表达,为进一步研究其生物学活性奠定了基础。Objective To construct recombinant Pichia pastoris secretion type expression vector and characterize immunogenicity of single chain variable fragment(scFv)of anti-idiotype monoclonal antibody against Vibrio alginolyticus.Methods Total RNA was extracted from hybridoma cell 2F4 secreting mAb against Vibrio alginolyticus,and then cDNA was amplified by RT-PCR.Then the RT-PCR product was recombined and inserted into secretion type expression vector pPIC9K-scFv.Recombinant vector was transformed into Pichia pasto...

关 键 词：溶藻弧菌 单链抗体 真核表达 毕赤酵母菌 

分 类 号：S941[农业科学—水产养殖]