摘要
目的:构建含有Hsvtk、重组人IL2rhIL2、TNFαrhTNFα不同组合融合基因的逆转录病毒表达载体。方法:经分离人外周血单个核细胞peripheralbloodmononuclearcellPBMC总RNA,以反转录聚合酶链反应reversetranscriptionpolymerasechainreactionRTPCR制备人IL2互补DNAcomplementaryDNAcDNA模板。分别以PCR方法扩增3种基因,扩增产物纯化后克隆至测序载体pGEMTEasy,挑取阳性克隆经鉴定后测序。将测序正确的各目的基因分别从测序载体相应酶切位点消化后,回收并纯化后与经相同双酶消化后的线性化PLXSN表达载体连接,连接产物常规转化大肠杆菌DH5α感受态细胞,经筛选后随机挑取阳性菌落后,提取质粒酶切鉴定插入片段大小和位置以获得正确重组子。按上述定向克隆方法依次将各片段正向插入至PLXSN表达载体的多克隆位点间,以构建含不同融合基因的真核表达载体。结果:经限制性酶切分析及PCR方法鉴定,各插入基因及融合基因大小、位置、方向均正确无误。结论:Hsvtk、IL2、TNFα不同组合融合基因表达载体的成功构建为喉癌的基因治疗提供部分实验基础也为表达具有多功能活性的新型融合蛋白和发现细胞因子的新功能提供有利条件。
Objective：To construct expressing ve ctors con taining different fusion genes of Hs v－tk，IL－２，and TNF－α．Metho ds ：The total RNA was extracted from huma n peripheral blood mononuclear cell（ PBMC）with isolation regents RNA zol R and the cDNA of human IL－２was synthesized through reverse transcription．T he three genes were amplified resp ectively by PCR and the purified PCR products were ligated into pGEM－T－Easy and transformed into JM１０９E．coli bacteria． The white clones were selected and the recombi nant plasmid was purified，which was further identified by restriction e ndonucleases and sequence．The recombina nt plasm id carrying correct gene and the retr oviral expressing vector PLXSN we re digested and transformed into DH５αE．coli bacteria．The clones were randoml y s elected and recombinant plasmid was purified，which was ientified by enzyme digestion again．By the methods of directional cloning the other sequenced genes were subcloned into corresponding restriction sites of PLXSN in turn to generat e different e ukaryotic expressing vector．Results：By the methods of restrictio n digestion and PCR we confirmed that the length，position and orientation of in serte d genes and fusion genes were all correct．Conclusion：The successful cons truction of expressing vectors containing diff erent fusion genes of Hsv－tk，IL －２，and TNF－αmay be beneficial for the gene therap y of laryngocarcinoma．It also can be use d for expressing new fusion proteins and studying novel biologi cal activities of cytokine．
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2001年第10期1019-1023,共5页
Chinese Journal of Cancer
基金
国家自然科学基金资助项目NO.39970788
