摘要
目的 构建嵌合跨膜型CD5 5基因的重组逆病毒表达质粒。方法 采用PCR技术扩增编码CD5 5信号肽及成熟蛋白胞外区 (-3 4 -3 0 4aa)的DNA片段 ,双侧引入EcoRⅠ、SspⅠ位点 ,与编码CD46分子的跨膜区及膜内区 (2 70～ 3 5 0aa)的DNA片段的5’末端自身的StuI位点连接 ,构建嵌合跨膜型CD5 5DNA片段 ,序列测定后将此嵌合CD5 5片段与pLXSN逆病毒载体连接构建重组表达质粒 ,酶切鉴定插入片段的方向。结果 DNA序列测定证实所构建的嵌合跨膜型CD5DNA阅读框完整、连接部位序列正确 ;HindⅢ酶切鉴定得到了正向插入的CD5 5TM pLXSN重组表达质粒。结论 我们成功构建了嵌合跨膜型CD5 5DNA片段及含此片段的重组逆病毒表达载体CD5 5TM pLXSN ,为进一步在真核细胞中表达嵌合分子 ,比较研究GPI锚固型CD5 5分子与细胞活化信号转导的关系建立良好对照并为应用跨膜型的CD5 5分子对PNH进行基因治疗奠定了分子生物学基础。
Objective To construct recombinant retrovirus vector of chimeric transmembrane form of CD55 gene. Methods Two primers P1, P2 were used to amplify the sequence of extracellular portion of CD55 by PCR. A Ssp I site was designed in the P2 to ligate with the Stu I site which was at the 5' end of the sequence of the transmembrane and cytoplasmic domains of MCP, so the transmembrane form of CD55(CD55 TM)gene was constructed. The chimeric CD55 DNA fragment was cloned into pGEM T Easy plasmid and sequenced, then the CD556 TM gene was ligated with the retrovirus vector pLXSN. Results The CD55TM DNA sequence was exactly consistent with our expectation, the open reading frame was right, and no frame shift, gene mutation; and digestion with HindⅢ showed that the gene had inserted into the retrovirus vector correctly. Conclusion We chimeric transmembrane form of CD55 gene have acquired its recombinant retrovirus vector constructed (CD55 TM pLXSN), which will be contribute to the investigation of the signal transduction of GPI form CD55 as on excellent control and provide a molecular biological basis for the new way for the gene therapy of PNH.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2001年第1期48-51,共4页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目! (3 963 0 2 96)
