反义B7—1质粒载体的构建与鉴定  

Construction and identification of eukaryotic expression vector of antisense B7-1

作　　者：李蕊[1] 米曰堂[1] 杨玉秀[2] 张颖慧[1] 

机构地区：[1]山东省聊城市人民医院普外科,252000 [2]山东省聊城市人民医院保健科,252000

出　　处：《中华实验外科杂志》2012年第8期1586-1588,共3页Chinese Journal of Experimental Surgery

摘　　要：目的构建反义B7—1质粒载体，为研究阻断B7—1／CD28共刺激分子通路提供基础。方法设计含有XhoI和BamHI酶切位点的1对引物，通过聚合酶链反应（PCR）技术扩增获取质粒载体pcDNA3．B7—1上的目的片段B7—1（118～530nt，426bp），克隆入PMDl8-T中间载体。PMD18-T-B7—1经XhoI和BamHI双酶切后，B7—1片段与pcDNA3B（-）连接，pcDNA3B（-）的XhoI和BamHI酶切位点与B7—1全基因序列所含酶切位点相反，构建反义B7．1表达载体。结果反义基因重组体经过酶切和PCR检测，得到426bp左有的目的基因片段。测序证明捅人的目的片段碱基序列与Genebank报道的B7-1基因序列（G1：111038144）中的B7—1片段100％同源，且反方向插入载体。结论成功构建大鼠B7—1反义真核表达载体，为阻断B7—1／CD28共刺激通路抑制排斥反应奠定了基础.Objective To construct plasmid vector of antisense-B7-1 which lays a good foundation for blockade of B7-1/CD28 passageway. Methods A pair of primers based on rat B7-1 gene with Xho I and BamH I restriction site were designed and synthesized. The fragment of BT-1 （118-530 nt, 426 bp） was amplified by polymerase chain reaction （PCR） from pcDNA3-B7-1 and cloned into the intermediary PMD18-T vector. BT-1 fragment was gained by enzyme digestion of Xbo I and BamH I, and ligated re- versely into the multiclone site of pcDNA3B（ - ） which clone site of BamH I and Xhol I was in an anti- sense orientation compared to BT-1. Results The expected gene fragment was about 426 base pairs by e- lectrophoresis and PCR method. A 426-bp fragment was in in accordance with the known B7-1 gene （ G1 ： 111038144）, and the insert direction was identified by DNA sequencing. Conclusion The recombinant plasmid pcDNA3B-BT-1 was constructed successfully. The availability of the pcDNA3B-BT-1 should facili- tate anti-rejection gene therapy in allograft transplantation.

关 键 词：共刺激分子B7—1 反义寡核苷酸类 真核表达载体 

分 类 号：R346[医药卫生—基础医学]