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目的探讨重组人β防御素2(hBD2)真核表达载体在人膀胱上皮细胞中的表达,并观察重组hBD2的体外抗菌活性。方法采用脂质体转染法将hBD2重组真核表达质粒pCAGG-hBD2导入无血清培养的人膀胱上皮细胞株T24细胞,利用RT-PCR法、Western blotting分别在核酸水平及蛋白质水平检测hBD2的表达。ELISA测定培养上清中hBD2的浓度,菌落计数法检测上清对尿路致病性大肠杆菌(UTI89)和克雷白杆菌(TOP52)的临床分离株的抗菌效果。结果 RT-PCR和Western blotting结果显示,转染后hBD2可在T24细胞中有效地表达。上清中hBD2的含量为(36.5±3.2)ng/106个细胞,菌落计数法显示,hBD2对UTI89和TOP52临床分离株有显著的杀菌作用。结论 hBD2重组真核表达载体脂质体法转染人膀胱上皮细胞后,可高效表达具抗菌活性的基因重组hBD2。
Objective To investigate the expression of eukaryotic expression vector of human β-defensin 2 (hBD2) in human bladder epithelial cells and to observe the in vitro antibacterial activity of recombinant human hBD2. Methods The hBD2 recombinant eukaryotic expression plasmid pCAGG-hBD2 was transfected into human bladder epithelial cell line T24 by using lipofection method. The expression of hBD2 was detected by RT-PCR and Western blotting respectively at the level of nucleic acid and protein . ELISA was used to determine the concentration of hBD2 in the culture supernatant. The colony counts were used to determine the antibacterial effect of the supernatant on the clinical isolates of UTI89 and TOP52. Results The results of RT-PCR and Western blotting showed that hBD2 could be efficiently expressed in T24 cells after transfection. The amount of hBD2 in the supernatant was (36.5 ± 3.2) ng / 106 cells, and the colony counting method showed that hBD2 had a significant bactericidal effect on the UTI89 and TOP52 clinical isolates. Conclusion The hBD2 recombinant eukaryotic expression vector liposome method transfected human bladder epithelial cells, high expression of antibacterial activity of recombinant hBD2.