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目的构建Na+,K+-ATP酶β1亚基(ATP1B1)真核表达质粒,为利用ATP1B1进行肿瘤基因治疗奠定基础。方法以白细胞文库为模板扩增ATP1B1 cDNA,定向插入到pEGFP-C3真核表达质粒中,构建pEGFP-ATP1B1重组质粒;经酶切分析和测序鉴定后,通过脂质体介导转染胃腺癌SGC-7901细胞,利用real-time PCR检测转染后SGC-7901细胞的ATP1B1基因表达,并进行其ATP酶活性检测;MTT法测定转染后SGC-7901细胞的增殖活性。结果重组质粒经鉴定证实含有ATP1B1 cDNA序列,转染pEGFP-ATP1B1的SGC-7901细胞ATP1B1基因表达增高达129.2%,ATP酶活性增强为(2.95±0.210)%,细胞生长增殖显著受抑。结论成功构建了ATP1B1真核表达质粒,为进一步利用ATP1B1研究恶性肿瘤基因治疗奠定基础。
Objective To construct the eukaryotic expression plasmid of Na +, K + -ATPase β1 subunit (ATP1B1) and lay a foundation for gene therapy of tumor with ATP1B1. Methods The leukocyte library was used to amplify ATP1B1 cDNA and inserted into the pEGFP-C3 eukaryotic expression plasmid to construct the recombinant plasmid pEGFP-ATP1B1. The recombinant plasmid was identified by restriction enzyme analysis and sequencing, and then transfected into gastric adenocarcinoma SGC -7901 cells. The expression of ATP1B1 gene in SGC-7901 cells was detected by real-time PCR and its ATPase activity was detected. The proliferation activity of SGC-7901 cells was determined by MTT assay. Results The recombinant plasmid was confirmed to contain the ATP1B1 cDNA sequence. The expression of ATP1B1 gene in SGC-7901 cells transfected with pEGFP-ATP1B1 increased by 129.2% and the ATPase activity increased by (2.95 ± 0.210)%. Cell proliferation was significantly inhibited. Conclusion The ATP1B1 eukaryotic expression plasmid was successfully constructed and laid the foundation for the further study on the gene therapy of malignant tumors by ATP1B1.