摘要
目的克隆与排异反应密切相关的iNOS基因cDNA片段,并构建反义转基因载体。方法培养人血管内皮细胞HVEC细胞株,采用反转录-PCR方法获取iNOS cDNA基因片段,并克隆入T载体并进行序列分析。用HindIII与BamHI双酶切带有iNOS cDNA片段的T载体质粒与pCDNA5载体,回收酶切产物,连接,转化,提取质粒并酶切鉴定。结果RT-PCR获取了669bp的iNOS基因片段;序列分析表明克隆的iNOS片段序列正确;双酶切分析显示克隆入pCDNA3载体的iNOS基因片段与预期的大小一致。结论克隆了iNOS基因片段,并构建了iNOS的反义转基因载体。
【Objective】 To clone rejection related iNOS cDNA fragment and construct antisense gene-transferring vector. 【Methods】 HVEC cell line was cultured. Total RNA was extracted out front HVEC cell lines, iNOS eDNA fragments were obtained by RT-PCR and cloned into T vector. The T vector with iNOS cDNA was sequenced, and cut by using HindⅢ and BamHI. The digested products were recycled and cloned into pcDNA5 vector, pcDNA5 with iNOS was extracted out and identified by enzyme digestion. 【Results】 669bp iNOS cDNA fragment was obtained by RT-PCR; T vector with iNOS fragment was identified by sequencing analysis; iNOS fragment length from pCDNA5 cut by Hind iii and BawH I was as expected. 【Conclusion】 iNOS fragment was successfully cloned, and its antisense gene-transferring vector was constructed successfully.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2006年第2期201-203,206,共4页
China Journal of Modern Medicine
关键词
INOS基因
排异反应
反义转基因载体
I NOS gene
rejection reaction
antisense gene-transferring vector
