鼠β-防御素2分泌性真核表达载体的构建及其稳定-第三军医大学学报.doc

鼠β-防御素2分泌性真核表达载体的构建及其Hepa1-6细胞稳定表达 梅寒芳1,2，金小宝，朱家勇*， （.广东药学院广东省药用生物活性物质重点实验室，广东 广州 510006; .南方医科大学热带医学与公共卫生学院, 广东 广州 510515摘要目的 克隆小鼠β防御素2（mBD2）基因,构建真核分泌性表达载体,Hepa 1-6细胞稳定表达。方法取C57BL/6肾总RNA,RT-PCR扩增mBD2成熟肽基因,Overlap-PCR在成熟肽基因上游加上鼠IgК信号肽基因，pcDNA3.1(+)，PCR、酶切和测序鉴定正确后，转染Hepa1-6细胞,G418筛选,RT-PCR和Western-Blot从mRNA和蛋白角度鉴定mBD2分子的表达。结果 成功信号肽IgК-mBD2序列，并且构建pcDNA3.1(+)/IgК-mBD2真核分泌性表达载体Hepa1-6细胞, RT-PCR从细胞总RNA中扩增得到202bp的IgК-mBD2基因，Western-Blot鉴定细胞培养上清中有mBD2蛋白分子表达。结论 成功构建pcDNA3.1(+)/IgК-mBD2真核分泌性表达载体,转染得到稳定表达mBD2的Hepa1-6细胞株,为进一步研究mBD2的抗肝癌免疫机制打下基础。Construction of murine beta-defensin 2 Secretory eukaryotic expressional vector and Identification of stable expressional Hepa1-6 cell line MEI Han-fang1,2, JIN Xiao-bao1,ZHU Jia-yong1*,ZENG Ai-hua1,WU Qiang1,LI Xiao-bo1,LU Xue-mei1, LIU Man-yu1 （.Guangdong key laboratory of pharmaceutical bioactive substances, Guangdong, Guangzhou 510006,China; 2.Institute of tropical medicine and public health, Southern medical university, Guangdong Guangzhou 510515,China） [Abstract] To clone murine beta defensin 2 (mBD2) gene from total RNA of mouse kidney, and to construct mBD2 secretary eukaryotic expressional vector, then realize its expression in Hepa1-6 cell. Methods Total RNA was isolated from kidney of C57BL/6 mice using TRIZOL reagent. DNA sequence coding for mature mBD2 was amplified by RT-PCR. To add a murine IgК signal peptide sequence using overlap-PCR. After successfully T-A clone, than subcloned IgК-mBD2 into the plasmid pcDNA3.1 (+).The pcDNA3.1 (+) / IgК-mBD2 was identified by PCR, endonuclease digestion, and sequencing. The Hepa1-6 cells were transfected with pcDNA3.1(+)/IgК-mBD2 using liposome2000 and selected with 400 mg /L G418 for 8 weeks. Identification of steady expression of mBD2 was confirmed by RT-PCR and Western-Blot. Results Successfully constructed the eukaryotic expression vector-pcDNA3.1 (+)/IgК-mBD2. Stable expressional cell line of Hepa1-6 was obtained by transfecting pcDNA3.1 (+)/IgК-mBD2 into Hepa 1-6 using Lipos