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目的:构建哺乳动物细胞表达的SPLUNC1重组蛋白,通过体外实验验证其杀菌/抑菌功能,并进一步研究其机制是否与SPLUNC1蛋白结合细菌脂多糖有关,从而为其将来临床应用奠定基础。方法:将SPLUNC1克隆入pCMV-tag4A哺乳动物表达载体,稳定转染鼻咽癌HNE1细胞株;收集细胞培养上清液,用其处理从患者上呼吸道分离的绿脓杆菌,比较转染SPLUNC1基因的细胞培养上清与转染空白载体的细胞培养上清对细菌克隆形成率的影响。同时将脂多糖包被96孔板,与SPLUNC1蛋白共同孵育,ELISA法检测SPLUNC1是否与细菌脂多糖结合;利用FITC标记的脂多糖干预转染SPLUNC1基因及空白载体的细胞,观察细胞内脂多糖与SPLUNC1的结合作用。结果:转染SPLUNC1基因的细胞培养上清能显著抑制绿脓杆菌在LB软琼脂板上形成集落;体外试验表明,SPLUNC1能与细菌脂多糖结合,但结合效率低;HNE1细胞经转染SPLUNC1后,细菌脂多糖摄取明显增加。结论:重组SPLUNC1蛋白能经转染细胞分泌到培养上清液中,具有结合细菌脂多糖,杀灭或抑制上呼吸道绿脓杆菌的功能,从而可能具有重要的临床应用价值。
OBJECTIVE: To construct SPLUNC1 recombinant protein expressed in mammalian cells and to verify its bactericidal / bacteriostatic activity in vitro and to further investigate whether its mechanism is related to the binding of SPLUNC1 protein to bacterial lipopolysaccharide, so as to lay the foundation for its future clinical application. Methods: SPLUNC1 was cloned into pCMV-tag4A mammalian expression vector and stably transfected into HNE1 cell line of nasopharyngeal carcinoma. The cell culture supernatants were collected and treated with Pseudomonas aeruginosa isolated from the upper respiratory tract of patients. The expression of SPLUNC1 gene Effect of cell culture supernatant and cell culture supernatant transfected with blank vector on bacterial colony formation rate. At the same time, lipopolysaccharide was coated on 96-well plate and incubated with SPLUNC1 protein. ELISA method was used to detect whether SPLUNC1 bound with bacterial lipopolysaccharide. Lipopolysaccharide was transfected into SPLUNC1 gene and blank vector by lipofectamine. SPLUNC1 binding. Results: The cell culture supernatant transfected with SPLUNC1 gene significantly inhibited the formation of colonies on LB soft agar plates. In vitro experiments showed that SPLUNC1 could bind to bacterial lipopolysaccharide but had low binding efficiency. After transfection of HNE1 cells into SPLUNC1 , Bacterial lipopolysaccharide uptake increased significantly. CONCLUSION: The recombinant SPLUNC1 protein can be secreted into the culture supernatant by transfected cells. It can bind bacterial lipopolysaccharide and kill or inhibit the function of Pseudomonas aeruginosa in upper respiratory tract, which may have important clinical value.