嵌合跨膜型人CD55基因的重组逆病毒表达质粒的构建

目的 构建嵌合跨模型CD55基因的重组逆病毒表达质粒。方法 采用PCR技术扩增编码CD155信号肽及成熟蛋白胞外区（-34-304aa)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（270～350aa)的DNA片段的5′未端自身的Stu I位点连接，构建嵌合跨模型CD55 DNA片段，序列测定后将此嵌合CD55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CD5 DNA阅读框完整、连接部位序列正确；Hind Ⅲ酶切鉴定得到了正向插入的CD55TM-pLXSN重组表达质粒。结论 我们成功构建了嵌合跨膜型CD55 DNA片段及含此片段的重组逆病毒表达载体CD55TM-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CD55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CD55分子对PNH进行基因治疗奠定了分子生物学基础。

Construction of recombinant retrovirus vector of chimeric transmembrane form of human CD55 gene

Objective To construct recombinant retrovirus vector of chimerictransmembrane form of CD55 gene. Methods Two primers P1, P2 were used to amplify the sequence of extracellular portion of CD55 by PCR. A SspI site was designed in the P2 to ligate with the StuI 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.