天然内生抗菌多肽elafin真核表达载体的构建及在真核细胞的表达

Construction of natural antibacterial endo-polypeptide elafin′s eukaryotic expression vector and its expression in eukaryotic cell

采用RT-PCR法提取天然内生抗菌多肽elafin的基因,通过测序确认后亚克隆构建真核表达载体pEGFP-N 1-elafin,再转染至肺上皮细胞A 549,荧光显微镜观察pEGFP-N 1-elafin在细胞中表达及定位,采用N orthern杂交、EL ISA检测法分别从转录及蛋白质水平分析表达情况,最后通过酶切电泳鉴定证实真核表达载体pEGFP-N 1-elafin构建成功,其转染细胞中有elafin的基因表达,且细胞上清液中elafin含量证实其主要是分泌性表达。E lafin基因cDNA真核表达载体的成功构建以及主要呈分泌性表达的特点显示了极具意义的潜在应用价值。

Objective To construct natural antibacterial endo-polypeptide elafin＇s eukaryotic expression vector, and observe its expression in pulmonary epithelial cell. Methods Total RNA was extracted from infected lung tissues, and elafin cDNA was obtained by RT-PCR. Its sequence was consistent with that of elafin provided by GenBank on internet, pEGFP-N1-elafin was constructed, and then transfected into pul- monary eithelial cell A549. The transcription and secretion of elafin were observe by fluorescence microscope and analyzed by Northern blotting and ELISA. Results Construction of pEGFP-Nl-elafin is successful, and elafin gene could be expressed in the transfected cells mainly in secretion form. Conclusion The eukaryotic expression vector of elafin has been constructed, and there is some evidence that the expression of elafin gene is mainly by early secretion in A549 which showed the potency of elafin in the treatment of chronic airway inflammation.