牛源整合素β1亚基的克隆及表达载体的构建.pdfVIP

2007年l2月 甘 肃 农 业 大 学 学 报 第 4 2卷 第 6期 18~22 JOURNAL OF GANSU AGRICULTURAL UNIVERSITY 双 月 刊 牛源整合素』3l亚基的克隆及表达载体的构建 杜 平 ，尚佑军 ，贺延玉。，孙晓林 ，马军武 (i．甘肃农业大学动物医学院，甘肃 兰州 730070；2．中国农业科学院兰州兽医研究所。 甘肃 兰州 730046；3．甘肃农业大学研究测试中心。甘肃 兰州 730070) 摘要：参照已公布整合素B】亚基的基因组序列，设计并合成了对克隆引物，以RT—PCR方法从牛肺组织扩增 出约2 400 bp的B 基因。经回收纯化连人PGEM--T载体。测序．依照测序结果设计i对原核表达引物。i对真核表 达引物，分别以克隆载体PGEMI3 为模板扩增目的片段，经相应的EcoR I、Xho I和EcoR I、Xba I酶切纯化后，在 T4DNA连接酶的作用下，定向亚克隆到原核表达载体pET一28a和真核表达载体 pCDNA3．1zero(+)中，PCR、酶 切鉴定及序列测定表明。成功构建了B 亚基原核表达载体 pETB-及真核表达载体 pCDNAI3 ． 关键词：口蹄疫病毒；整合素；B-亚基；表达载体 中图分类号：S 823：Q785 文献标识码：A 文章编号：1003—4315(2007)06—0018—05 Cloning of bovine integrin J3l gene and construction of its expression vector DU Ping 。SHANG You—jun。，HE Yan—yu。，SUN Xiao—lin 。MA Jun—WU。 (1．College of Veterinary Medicine，Gansu Agricultural University，Lanzhou 730070，China；2．Lanzhou Veterinary Research Institute。Chinese Academy of AgriculturaI Science，Lanzhou 730046，Chin；3．InstrumentaI Research and Analysis Center．Gansu Agricultural University，Lanzhou 730070，China) Abstract：Two pairs of specific primers referring to the bovine integrin betal gene in Genebank were designed and synthesized，a 2400 bp fragment was produced by reverse transcription polymerase chain reac— tion(RT—PCR)from bovine lung．The amplified fragment was cloned to pGEM ——T easy vector．con- firmed by sequenceing．Then a pair of prokaryotic and euokaryotic expression primers was designed on the basis of sequenceing results．The expression sequences from the cloned vector pGEMbetal were amplified and digested respectively by the restricted enzyme EcoR I、Xho I and EcoR I、Xba I．The digested and puri- fied betal gene of bovine were cloned into pr