重组真核表达载体pEGFP—C1／cecropin—XJ的构建及其在胃癌细胞MGC80—3中的表达

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—3，48h后观察EGFP瞬时表达情况；72h后，应用RT—PCR，检测抗菌肽cecropin—XJ基因的表达；96h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48h后，荧光显微镜下可观察到EGFP的表达，发出绿色荧光；转染72h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．

Construction of Recombinant Eukaryotic Expressive Vector pEGFP-C1/Cecropin-XJ and Its Expression in Gastric Cancer Cells MGC80-3

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.