摘要
目的改构α-防御素-1(HNP-1)使其成为一端带J链的改构体J-HNP-1分子,并探索建立能有效表达和分泌J-HNP-1,其表达产物又易于被检测和分离纯化的哺乳动物细胞表达系统。方法利用重组PCR技术,使HNP-1一端带上J链;将J-HNP-1 DNA片段插入哺乳动物细胞表达载体pcDNA3.1(-)/Myc-HisC中,构建出C端带Myc和6×His的rpcDNA3.1(-)/Myc-HisC/J-HNP-1;将此重组真核表达载体导入COS-7细胞,分别从mRNA和蛋白质水平分析J-HNP-1的表达情况,并同步检测细胞可溶性蛋白及培养上清的抗菌活性。结果采用RT-PCR法从被转染的细胞中扩增出1条约786bp的片段,其大小与预测相符;采用Western印迹法,用抗His抗体检测到细胞可溶性蛋白及培养上清在相对分子质量约24×103处有强反应条带显示;细胞可溶性蛋白及培养上清的抗菌活性检测显示,经rpcDNA3.1(-)/Myc-HisC/J-HNP-1转染的COS-7细胞的可溶性蛋白和培养上清均有明显的抗菌活性。结论α-防御素-1(HNP-1)被成功改构成杀菌肽J-HNP-1;所建立的J-HNP-1哺乳动物细胞表达载体系统能有效表达和分泌活性J-HNP-1。
OBJECTIVE To reconstruct the HNP-1 into the J-HNP-1 with a J chain, and explore to set up a mammalian cell expression system which can express and secret J-HNP-1, so that the products could be examined and purified conveniently. METHODS The J-HNP-1 cDNA fragments were produced by recombinant PCR. Then the J-HNP-1 was inserted into the mammalian expression vector pcDNA3.1 （-）/Myc-His which had the double marks Myc and 6× His. The recombinant vector rpcDNA3.1 （-）/Myc-His /J-HNP-1 was transfected into the COS-7 cell. The J-HNP-1 expression was analyzed at the mRNA and protein level. The germicidal activity of cell culture supernatant and cellular solution protein was assayed. RESULTS By the use of RT-PCR with special primers, a band of 786bp was amplified from COS-7 cells transfected by this recombinant plasmid. Western blot analysis with specific anti-histidines antibody revealed that the cell culture supernatant and cellular solution protein of COS-7 cells transfected by rpcDNA3.1 （-）/Myc-His/J-HNP-1 had a strong band with relative molecular mass of about 24 × 10^3. Antibacterial activity assay showed that obvious bacterial inhibition occurred in both lysate and supernatant of COS-7 cells transfected by rpcDNA3.1 （ - ）/Myc-His /J-HNP-1. CONCLUSIONS The J-HNP-1 recombinant is obtained and inserted into the mammalian expression vector then to be expressed in vitro. The expression product is found to have the anti-bacterial effect in vitro.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2006年第2期129-133,共5页
Chinese Journal of Nosocomiology
基金
国家自然科学基金项目(30100197)
