鸡氨肽酶N蛋白多克隆抗体制备及其生物学功能研究  

Preparation of polyclonal antibody against chicken amino peptidase N protein and its biological functions

作　　者：李广兴[1] 李兰兰[1] 潘龙[1] 洪琴[1,2] 张恒[1,3] 杨巍[1] 黄小丹[1,4] 马德星[1] 张瑞莉[1] 杨贵君[1] 

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030 [2]上海药明康德新药开发有限公司生物部,上海200131 [3]山东信得科技股份有限公司,山东潍坊262200 [4]黑龙江职业学院,哈尔滨150080

出　　处：《东北农业大学学报》2015年第2期24-31,共8页Journal of Northeast Agricultural University

基　　金：国家自然科学基金项目(31172295;31272569);黑龙江省自然科学基金项目(ZJN0702-01)

摘　　要：试验参考Gen Bank上鸡氨肽酶N(g APN)基因序列(登录号为NM_204861.1)设计多对特异性引物,利用RT-PCR分段克隆g APN基因各段克隆产物并利用SOE-PCR将其进行连接,得到大小为2 906 bp的全长基因。利用原核表达载体p ET-30a(+)构建重组质粒p ET-g APN并转化E.coli Rosetta,经诱导表达得到大小为113 ku目的条带。以纯化g APN重组蛋白为免疫原制备兔抗g APN多克隆抗体,间接Elisa方法检测多抗血清效价为215。Western Blot试验证明,此多克隆抗体可以与原核表达的蛋白产生特异性条带,同时与18日龄鸡肾组织中获得的天然g APN蛋白样品有良好的反应性。间接免疫荧光试验显示,多克隆抗体可检测到pc DNA-g APN转染HELA细胞所表达的g APN蛋白。上述结果可为g APN蛋白的深入研究奠定基础。In this study, specific primers were designed according to the reference g APN gene（NM_204861.1） of Gen Bank. The complete chicken amino peptidase N（g APN） gene of totally 2 906 bp was cloned and linked with RT- PCR and SOE PCR technique. The g APN was expressed after construction of p ET- 30a- g APN and transformed E. coli Rosetta, the molecular weight of g APN recombinant protein is 113 ku. The purified recombinant protein was used as antigen for preparation of rabbit anti- g APN polyclonal antibody, the titer of polyclonal antibody was 215. Western Blot showed that this polyclonal antibody had highly reactivity and specialty with recombinant protein and natural g APN from kidney of 18-day-old of chicken. IFA test demonstrated that this polyclonal antibody could react with the HELA cell transfected with eukaryotic expression plasmid of pc DNA-g APN.

关 键 词：鸡氨肽酶N 多克隆抗体 免疫印迹 间接免疫荧光 

分 类 号：S852.23[农业科学—基础兽医学]