双靶向调控CDX2基因慢病毒表达载体的构建及鉴定

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-5HRE-hTERTp-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-5HRE-hTERTp、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。

Construction and Identification of Dual Target-Regulated Lentiviral Vector of Colorectal Cancer Suppressor Gene CDX2

Objective To build a lentiviral expression vector regulated by two targets 5 copies of HREs and hTERTp, express the target gene CDX2, and to test the activity of hTERT promoter by using LoVo cells for transfection. Methods After the primer sets were designed, the hTERT promoter was cloned by PCR amplification from the genome of colon cancer. The CEA promoter was removed from the original vector pLEGFP-5HRE-CEAp by double digestion and PCR method, and then the hTERTp was introduced into the vector to construct the recombinant plasmid pLEGFP-5HRE-hTERTp. 5HRE-hTERTp was obtained by PCR, while the CMV promoter was removed from the original vector pLVX-EGFP-3FLAG by double digestion and PCR method, and then the 5HRE-hTERTp was introduced into the vector to construct the recombinant plasmid pLVX-5HRE-hTERTp-EGFP-3FLAG. The CDX2 was cloned by PCR amplification from GV230-CDX2-EGFP, and the EGFP was removed from the vector pLVX-5HRE-hTERTp-EGFP-3FLAG by double digestion, and then the CDX2 was introduced into the vector to construct the recombinant plasmid pLVX-5HRE-hTERTp-CDX2-3FLAG. LoVo cells ex vivo was transiently transfected by pLVX-5HRE-hTERTp-EGFP-3FLAG to evaluate the activity of hTERTp by detecting the expression of green fluorescence protein EGFP. Results PCR and sequencing analyzing showed that pLEGFP-5HRE-hTERTp, pLVX-5HRE-hTERTp-EGFP-3FLAG, and pLVX-5HRE-hTERTp-CDX2-3FLAG were sequenced correctly and the same as our designed. pLVX-5HRE-hTERTp-EGFP-3FLAG was successfully transfected into LoVo cells ex vivo and expressed green fluorescence protein EGFP, which showed that hTERTp was activated and promoted the expression of downstream gene. Conclusion The lentiviral expression vector, pLVX-5HRE-hTERTp-EGFP-3FLAG and pLVX-5HRE-hTERTp-CDX2-3FLAG are successfully constructed, which lays the foundation of further research. But the function of dual-target regulation needs further proof.