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目的构建5个拷贝的HRE和hTERTp双靶向调控表达CDX2基因的慢病毒表达载体,并在结肠癌LoVo细胞中检测其启动活性。方法设计引物应用PCR法从人结肠癌基因组中克隆获得hTERT启动子,用双酶切和PCR法切除笔者所在课题组已构建的载体pLEGFP-5HRE-CEAp中的CEA启动子后,将hTERT启动子与该载体重组,构建出pLEGFP-5HRE-hTERTp;提取5HRE-hTERTp基因序列,同时双酶切切除pLVX-EGFP-3FLAG载体中的CMV启动子,同源重组构建获得pLVX-5HRE-hTERTp-EGFP-3FLAG载体。设计引物应用PCR法从笔者所在课题组已构建的GV230-CDX2-EGFP载体中克隆获得CDX2基因序列,用双酶切和PCR法切除载体pLVX-5HRE-hTERTp-EGFP-3FLAG中的EGFP后,将CDX2基因序列与该载体重组,构建出pLVX-5HREhTERTp-CDX2-3FLAG。应用pLVX-5HRE-hTERTp-EGFP-3FLAG载体瞬时转染体外培养的人结肠癌LoVo细胞,通过观察绿色荧光蛋白EGFP的表达,鉴定hTERTp的启动活性。结果经PCR和测序分析,pLEGFP-5HREhTERTp、pLVX-5HRE-hTERTp-EGFP-3FLAG和pLVX-5HRE-hTERTp-CDX2-3FLAG 3组质粒与设计一致,测序正确。将pLVX-5HRE-hTERTp-EGFP-3FLAG载体瞬时转染体外培养的人结肠癌LoVo细胞,有绿色荧光表达,提示hTERT启动子能够有效启动下游基因的表达。结论成功构建了pLVX-5HRE-hTERTp-EGFP-3FLAG及pLVX-5HRE-hTERTp-CDX2-3FLAG载体,为后续的体外和体内研究打下了基础。
Objective To construct five copies of lentiviral vector expressing CDX2 gene targeting HRE and hTERTp and to detect its promoter activity in colon cancer LoVo cells. Methods Primer was designed to clone hTERT promoter from human colon cancer genome by PCR. The CEA promoter in pLEGFP-5HRE-CEAp vector was excised by double enzyme digestion and PCR. The hTERT promoter The recombinant plasmid pLVX-5HRE-hTERTp-EGFP-EGFP-3FLAG was constructed by recombination with the vector to construct pLEGFP-5HRE-hTERTp. The 5HRE-hTERTp gene sequence was extracted and the CMV promoter in pLVX- 3FLAG vector. CDX2 gene was cloned from the GV230-CDX2-EGFP vector constructed by the author’s group by PCR method. The EGFP in vector pLVX-5HRE-hTERTp-EGFP-3FLAG was excised by double enzyme digestion and PCR The CDX2 gene sequence was recombined with this vector to construct pLVX-5HREhTERTp-CDX2-3FLAG. The human colon cancer LoVo cells were transiently transfected with pLVX-5HRE-hTERTp-EGFP-3FLAG vector and the activation of hTERTp was identified by observing the expression of green fluorescent protein EGFP. Results PCR and sequencing analysis, pLEGFP-5HREhTERTp, pLVX-5HRE-hTERTp-EGFP-3FLAG and pLVX-5HRE-hTERTp-CDX2-3FLAG 3 plasmid consistent with the design and sequencing correctly. Transient transfection of pLVX-5HRE-hTERTp-EGFP-3FLAG vector into human colon cancer LoVo cells showed green fluorescent expression, suggesting that hTERT promoter can effectively activate downstream gene expression. Conclusion The pLVX-5HRE-hTERTp-EGFP-3FLAG and pLVX-5HRE-hTERTp-CDX2-3FLAG vectors were successfully constructed, which laid the foundation for subsequent in vitro and in vivo studies.