带J链的α-防御素-1基因转染COS-7细胞后的表达检测

目的将α-防御索-1（HNP-1）基因与J链基因重组为一种新的杀菌肽分子J-HNP-1，观察J-HNP-1在细胞内的表达及分泌情况。方法采用聚合酶链反应（PCR）方法分别从pCH-J质粒和pBabeNco-HNP-1质粒中扩增出J链和HNP-1；进行重组PCR，获取J-HNP-1 DNA片段．并插入哺乳动物细胞表达载体pcDNA3．1（-）／Myc-HisC中；用脂质体转染法将此重组真核表达载体导入COS-7细胞。然后应用蛋白质免疫印迹法（Western blot）检测His-tag标签基因。结果用抗His抗体检测到细胞可溶性蛋白及培养上清在约24ku处均有强反应条带显示，其大小与预测相符；对照组未见相应条带显示。结论采用Western blot检测到新的杀菌肽分子J-HNP-1在细胞内得到表达并分泌到细胞外．为进一步研究J-HNP-1杀菌肽的抗菌活性奠定了基础。

Expression of -defensin-1 with J chain in the transfected COS-7 cells

OBJECTIVE: To recombine the alpha-defensin-1 (HNP-1) and J chain cDNA fragment into a new germicidal molecule J-HNP-1, and investigate its expression in the transfected COS-7 cells. METHODS: J chain and HNP-1 cDNA were amplified from the plasmids pCH-J and pBabeNeo-HNP-1 respectively, then the J-HNP-1 DNA fragment was obtained by recombinant polymerase chain reaction (PCR). The J-HNP-1 fragments were inserted into pcDNA3.1 (-)/Myc-HisC, which subsequently was made to transfect the COS-7 cell by lipid transfection method. The expression was confirmed by Western blot. RESULTS: Determination with His antibody, a dense band at 24 ku was displayed by the cell lysate and the cell culture supernatant in concordance with that of the predicted extent, but not in the control. CONCLUSION: The new germicidal peptide J-HNP-1 is found to be expressed intracellularly and could be secreted. The results lay the foundation for further study on the germicidal activity of J-HNP-1.