摘要
目的构建新疆家蚕抗菌肽(cecropin-XJ)基因的真核表达载体pEGFP-C1/cecropin-XJ(pEGFP-cec),检测其在肿瘤细胞中的表达情况,探讨抗菌肽对肿瘤细胞的作用机制和抗菌肽抑瘤作用效果.方法应用基因重组技术,将新疆家蚕抗菌肽(cecropin-XJ)基因克隆到真核表达载体pEGFP-C1,通过酶切和测序的方法鉴定重组质粒pEGFP-cec的正确性.将pEGFP-cec经脂质体法转染到胃癌细胞MGc80-3,48 h后观察EGFP瞬时表达情况;72 h后,应用RT-PCR,检测抗菌肽cecropin-XJ基因的表达;96 h后,消化细胞,经台盼蓝染色,检测重组质粒pEGFP-cec表达产物对肿瘤细胞的影响.结果细胞转染48 h后,荧光显微镜下可观察到EGFP的表达,发出绿色荧光;转染72 h后,RT-PCR检测到胞内有抗菌肽cecropin-XJ基因的表达;表达产物能够抑制肿瘤细胞的生长.结论成功构建了真核表达载体pEGFP-cec,并在肿瘤细胞中可见抗菌肽cecropin-XJ和EGFP基因的有效表达以及表达产物具有显著的抗肿瘤活性.为深入开展抗菌肽抑制肿瘤的研究奠定基础.
Purpose to construct a eukaryotic expressive vector with cecropin-XJ gene pEGFP-C1/cecropin-XJ （pEGFP-cec）, to detect expression of cecropin-XJ gene in tumor cell and to investigate its related function in cells and the mechanism of inhibiting tumor. METHODS： The cecropin-XJ gene from pcDNA3.1/cecropin-xj was cloned into vector pEGFP-C1 and sequenced. The recombinant vector was transfected into gastric cancer cells MGC80-3 with lipofectin. The EGFP expression was observed by fluorescence microscropy 48 h after transfection and the cecropin-xj mRNA expression was detected by RT-PCR 72 h after transfection. The effect of expression produces on MGC80-3 cells was valued by trypan blue dye Method. RESULTHS： The vector pEGFP-cec was successfully constructed ; the expression of EGFP was observed in the fluorescence microscropy 48 h after transfection, and the green fluorescence; cecropin-xj mRNA was detected by RT-PCR 72 h after transfection and the expression produces of recombinant vectors could inhibit the growth of tumor cells. CONCLUSION ： The eukaryotic expression vector pEGFP-cec was constructed successfully and cecropin-XJ and EGFP gene could effectively be expressed in tumor cells the expression produces is active, which providedes support for tumor gene therapy of antibacterial peptide.
出处
《新疆大学学报（自然科学版）》
CAS
2007年第4期449-453,共5页
Journal of Xinjiang University(Natural Science Edition)
基金
自治区高技术研究发展计划项目(200311118)
新疆高校创新研究群体基金项目(XJEDU2004G02)
