J-HNP-1多肽的重组及体外抗菌作用初步研究

发布时间：2018-11-19 19:27

【摘要】：条件致病菌感染病原菌多为耐药菌,其主要来源于呼吸道、肠道、生殖道等粘膜表面,其感染常见于严重创伤、烧伤、移植术后、成人免疫缺陷综合征等重症患者。目前尚无良好的预防手段。粘膜上皮细胞有一个共同特点,即在细胞膜上具备多聚IgA受体(pIgR)。连有J链的IgA分子通过J链与pIgR结合后,将IgA分子转运入粘膜细胞内。本研究拟将防御素改建为一种杀菌分子,分子的一端为J链,另一端为防御素,称之为多聚IgA受体(pIgR)配体样杀菌肽。这种杀菌分子因连有J链,具有与pIgR结合的能力。从而利用pIgR作为桥梁,将防御素转运至细胞内的微生物感染处发挥杀菌作用。本研究将HNP-1(属α-防御素)与J链cDNA连接,然后在哺乳动物细胞系统中表达,并初步检测其产物体外抗菌活性,为进一步研究其细胞内杀菌功能提供依据。方法: 应用PCR技术从不同的质粒中获得J链基因和HNP-1基因,然后应用重组PCR技术将这两个不同的基因连接在一起而成为J-HNP-1基因。将此基因克隆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子导入COS-7细胞,分别从mRNA和蛋白质水平采用RT-PCR和Western blot分析J-HNP-1的表达情况,并体外检测细胞可溶性蛋白及培养上清的抗菌活性。结果: 1.利用重组PCR技术使J链与HNP-1这两个不同的基因相连接而成为J-HNP-1重组体。 2.将J-HNP-1重组体克隆入哺乳动物表达载体pcDNA3.1(-)/Myc-HisC质粒中,双重标签基因Myc和6×His位于下游,6×His起着检测标志的作用,使J-HNP-1的表达产物易于检测。 3.将重组质粒rpcDNA3.1(-)/Myc-HisC/J-HNP-1应用正、反方向引物测序,检测J-HNP-1的DNA序列及鉴定插入的正反方向,获得正向插入重组子。 4.RT-PCR结果显示转染rpcDNA3.1(-)/Myc-HisC/J-HNP-1的COS-7细胞mRNA
[Abstract]:Most of the pathogenic bacteria are drug-resistant bacteria which mainly come from the mucosal surface of respiratory tract intestinal tract and genital tract. The infection is common in severe patients such as severe trauma burn transplantation adult immunodeficiency syndrome and so on. At present, there are no good preventive measures. A common feature of mucosal epithelial cells is the presence of a poly-IgA receptor (pIgR). On the cell membrane. The IgA molecule with J chain transports IgA molecules into mucosal cells after binding to pIgR via J chain. In this study, defensin was transformed into a bactericidal molecule with a J-chain at one end and a defensin at the other end, which was called a poly (IgA receptor) ligand-like bactericidal peptide (pIgR). This bactericidal molecule has the ability to bind to pIgR because of its J chain. Using pIgR as a bridge, defensins can be transported to microbe infection to play a bactericidal role. In this study, HNP-1 (伪 -defensin) was linked to J chain cDNA, then expressed in mammalian cell system, and its antibacterial activity in vitro was preliminarily detected, which provided the basis for further study of its bactericidal function in cells. Methods: J chain gene and HNP-1 gene were obtained from different plasmids by PCR technique, then the two genes were linked together by recombinant PCR technique to become J-HNP-1 gene. The gene was cloned into mammalian cell expression vector pcDNA3.1 (-) / Myc-HisC. The recombinant plasmid was transfected into COS-7 cells by liposome transfection. The expression of J-HNP-1 was analyzed by RT-PCR and Western blot from mRNA and protein levels, and the antibacterial activity of soluble protein and culture supernatant was detected in vitro. Results: 1. By using recombinant PCR technique, J chain was linked to two different genes of HNP-1 to form J-HNP-1 recombinant. 2. The J-HNP-1 recombinant was cloned into the mammalian expression vector pcDNA3.1 (-) / Myc-HisC. The double label genes Myc and 6 脳 His were located downstream, and 6 脳 His acted as a detection marker, making the expression product of J-HNP-1 easy to detect. 3. The recombinant plasmid rpcDNA3.1 (-) / Myc-HisC/J-HNP-1 was sequenced with positive and reverse primers to detect the DNA sequence of J-HNP-1 and to identify the positive and negative directions of the insertion. 4.RT-PCR results show mRNA of COS-7 cells transfected with rpcDNA3.1 (-) / Myc-HisC/J-HNP-1
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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