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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.
Objective To construct the eukaryotic expression vector pEGFP-C1 / cecropin-XJ (pEGFP-cec) of the cecropin-XJ gene in Xinjiang silkworm (Bombyx mori) to detect the expression of the cecropin-XJ gene in tumor cells and to explore the mechanism of action of the antimicrobial peptide on tumor cells. Anti-tumor effect of antimicrobial peptides.Methods The gene of cecropin-XJ was cloned into eukaryotic expression vector pEGFP-C1 by gene recombination technology, and the correctness of recombinant plasmid pEGFP-cec was identified by restriction enzyme digestion and sequencing The transfection of pEGFP-cec into gastric cancer cell MGc80,3,48 h after transfection was used to observe the transient expression of EGFP. After 72 h, the expression of cecropin-XJ gene was detected by RT-PCR. After 96 h , The cells were digested and stained with trypan blue to detect the effect of the recombinant plasmid pEGFP-cec on the tumor cells.Results The expression of EGFP was observed under a fluorescence microscope 48 h after transfection, The expression of cecropin-XJ gene was detected by RT-PCR and the expression of cecropin-XJ gene was inhibited by RT-PCR.Conclusion The eukaryotic expression vector pEGFP-cec was successfully constructed and the antitumor peptide cecropin- Efficient expression of XJ and EGFP genes Expression product has significant anti-tumor activity. Peptide lay the foundation for depth of tumor suppression.