中国林蛙抗菌肽Temporin-1CEa基因的真核表达载体构建。

摘要

In order to establish a method to get a large number of antimicrobial peptides from Rana chensinensis,a series of experiments were conducted as follows.According to Chinese frog skin antimicrobial peptides Temporin-1CEa gene mRNA sequence（EU624139） in GenBank,a pair of specific primers were designed and cDNA was obtained from Chinese forest frog skin RNA by reverse transcription.Temporin-1CEa gene coding sequence was amplified using the cDNA,and linked with pEASY-T3 cloning vector.The GFP gene was inserted into the recombinant plasmid Tem-T3 by molecular methods.The Tem-GFP fragment was linked with eukaryotic expression vector pcDNA3.1,and Tem-GFP-pcDNA3.1 recombinant plasmid was achieved finally.Using of lipid infection method,the plasmids were transfected into sheep fibroblast cells,the green fluorescence was observed under a fluorescence microscope after 48 h.qPCR data showed that Tem-GFP fusion protein expression level of transfected Tem-GFP-pcDNA3.1 sheep fibroblasts increased about 300 folds than that of the control group.This study supplied the technical basis for developing mammary gland bioreactor of expressing Temporin-1CEa gene.%为了建立大量获取中国林蛙抗菌肽的方法,根据GenBank中的中国林蛙皮肤抗菌肽Temporin-1CEa基因的mRNA序列（EU624139）设计一对特异性引物,以提取的中国林蛙皮肤总RNA反转录出的cDNA为模板,将扩增的编码序列与pEASY-T3克隆载体连接获得Tem-T3;利用酶切、连接等分子生物学手段,将GFP基因连入Tem-T3克隆载体,再经酶切获得Tem-GFP片段,并插入真核表达载体pcDNA3.1,最终得到Tem-GFP-pcD-NA3.1重组质粒;利用脂质体转染法将该质粒转入绵羊成纤维细胞,48 h后可在荧光倒置显微镜下观察到GFP的绿色荧光表达;qPCR数据分析显示,与对照组相比,转染Tem-GFP-pcDNA3.1的绵羊成纤维细胞中融合蛋白Tem-GFP的表达量可提高约300倍。本研究为构建Temporin-1CEa基因山羊乳腺特异表达载体提供依据。