成纤维细胞激活蛋白及其突变体真核表达质粒的构建及鉴定

摘要

Objective To construct and identifythe eukaryotic expression plasmids containing wild -type FAPαand its mutants .Methods The wFAPαsequence and tFAPαsequence obtained by RT -PCR amplification using a high fidelity gene expression profiling strategy mediated by Pfu proofreading polymerases , were cloned into the eukaryotic ex-pression vector pcDNA3.1(+).The mFAPαwas obtained by overlap PCR using the pcDNA -wFAPαas template and cloned into the same vector .Results The construction of pcDNA-wFAPα, pcDNA-tFAPαand pcDNA-mFAPαwas confirmed by enzyme digestion and DNA sequencing .Conclusion The eukaryotic expression plasmids have been con-structed successfully , which lays important foundation for the future research on the molecular mechanism of FAP αin oral squamous cell carcinoma carcinogenesis .%目的：构建并鉴定人成纤维细胞激活蛋白（ FAPα）真核表达质粒pcDNA－wFAPα（野生型FAPα）、pcDNA－tFAPα（胞外段FAPα）和pcDNA－mFAPα（缺失624～704位氨基酸的突变型FAPα）。方法以口腔癌组织总RNA为模板，采用RT－PCR方法扩增wFAPαcDNA基因片段，连接至真核表达质粒pcDNA3．1（＋）获得重组pcDNA－wFAPα质粒。以重组 pcDNA －wFAPα为模板，扩增 tFAPα基因片段；用 overlap PCR 技术，获得mFAPα基因；分别连接至pcDNA3．1（＋）获得重组pcDNA－tFAPα和pcDNA－mFAPα质粒。结果酶切鉴定和测序验证皆显示pcDNA－wFAPα、pcDNA－tFAPα和pcDNA－mFAPα为正确重组质粒。结论成功构建人野生型FAPα（ wFAPα）、胞外段FAPα（ tFAPα）和突变型FAPα（ mFAPα）真核表达质粒，为进一步研究FAPα在口腔鳞癌发病过程中的分子机制奠定基础。