CAJ | 학술논문

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Xho Ⅰ和BamH Ⅰ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～ 530 nt,426 bp),克隆入PMD18-T中间载体.PMD18-T-B7-1经Xho Ⅰ和BamH Ⅰ双酶切后,B7-1片段与pcDNA3B(-)连接,pcDNA3B(-)的Xho Ⅰ和BamH Ⅰ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426 bp左右的目的基因片段.测序证明插入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.
목적 구건반의B7-1질립재체,위연구조단B7-1/CD28공자격분자통로제공기출.방법 설계함유Xho Ⅰ화BamH Ⅰ매절위점적1대인물,통과취합매련반응(PCR)기술확증획취질립재체pcDNA3-B7-1상적목적편단B7-1( 118～ 530 nt,426 bp),극륭입PMD18-T중간재체.PMD18-T-B7-1경Xho Ⅰ화BamH Ⅰ쌍매절후,B7-1편단여pcDNA3B(-)련접,pcDNA3B(-)적Xho Ⅰ화BamH Ⅰ매절위점여B7-1전기인서렬소함매절위점상반,구건반의B7-1표체재체.결과 반의기인중조체경과매절화PCR검측,득도426 bp좌우적목적기인편단.측서증명삽입적목적편단감기서렬여Genebank보도적B7-1기인서렬(G1:111038144)중적B7-1편단100％동원,차반방향삽입재체.결론 성공구건대서B7-1반의진핵표체재체,위조단B7-1/CD28공자격통로억제배척반응전정료기출.
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 B7-1 ( 118-530 nt,426 bp) was amplified by polymerase chain reaction (PCR) from pcDNA3-B7-1 and cloned into the intermediary PMD18-T vector.B7-1 fragment was gained by enzyme digestion of Xho Ⅰ and BamH I,and ligated reversely into the multiclone site of pcDNA3B( - ) which clone site of BamH I and Xhol Ⅰ was in an antisense orientation compared to B7-1.Results The expected gene fragment was about 426 base pairs by electrophoresis 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-B7-1 was constructed successfully.The availability of the pcDNA3B-B7-1 should facilitate anti-rejection gene therapy in allograft transplantation.