绵羊Nectin 4基因的克隆与表达  

Cloning and expression of sheep Nectin 4 gene

作　　者：王秋霞[1,2] 郭利亚[3,4] 陈蕾[2] 窦永喜[2] 欧长波[1] 余燕[1] 张艳红[1] 马金友[1] 刘兴友[1] 才学鹏[2] 

机构地区：[1]河南科技学院动物科学学院,河南新乡453003 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046 [3]甘肃农业大学动物科学技术学院,甘肃兰州730070 [4]中国农业科学院北京畜牧兽医研究所动物营养国家重点实验室,北京100193

出　　处：《中国兽医科学》2016年第1期116-121,共6页Chinese Veterinary Science

基　　金：现代农业产业技术体系建设专项(CARS-40-10);公益性行业(农业)科研专项经费项目(201103008);河南省高等学校重点科研项目(15A230002)

摘　　要：采用巢式PCR方法获得未见报道的绵羊Nectin4基因。根据GenBank上登录的牛的Nectin4mRNA序列信息设计并合成10对引物;从绵羊肺支气管组织中提取总RNA,采用随机引物和Oligo(dT)将其反转录为cDNA;利用巢式PCR原理和PCR引物错配导致非特异反应的特点,改变常规PCR反应条件,将10对引物分为2组,通过两轮PCR即获得大小片段相符的片段;TA克隆后测序证实已成功获得绵羊Nectin4基因的完整ORF,证明本方法可作为一种获取未知基因的方法,较RACE等其他传统技术有明显优势;构建了重组表达质粒pCMV-Myc-Nectin 4,在CHO细胞的细胞质中获得高效表达,经Western-blot分析,表达的Nectin 4蛋白的大小与预期符合。绵羊Nectin4基因的成功扩增为后续对其在病毒入侵中的功能进行研究奠定了基础。Nested PCR was applied to amplify the unreported Nectin 4gene of sheep.Ten pairs of primers were designed based on bovine Nectin4 mRNA in GenBank.Total RNA was extracted from bronchial tissue of sheep lungs,and then reverse-transcribed to cDNA using random primer and Oligo（dT）primer.According to the principle of nested PCR and the nonspecific reactions caused by the PCR primers mismatches,adjust PCR reaction conditions and divide these ten pairs of primers into two groups.The fragment with suitable size was finally obtained through two rounds of PCR.The complete ORF of sheep Nectin 4was confirmed by sequencing followed TA cloning.This study proved that this method can be used to obtain Nectin 4gene of sheep and displayed obvious advantage compared to the traditional methods such as RACE.Recombinant expression plasmid pCMV-Myc-Nectin 4was constructed.It was highly expressed in CHO cells.The size of Nectin 4protein expressed in the CHO cells was analyzed and confirmed by Western-blot.The successful amplification of sheep Nectin4 gene lays the foundation for the further study of the function in virus invasion.

关 键 词：Nectin4 巢氏PCR 载体构建 蛋白质表达 

分 类 号：S852.659.5[农业科学—基础兽医学]