摘要
目的 分离和鉴定刺激人肺腺上皮细胞 (SPC A 1) β 防御素 1(hBD 1)基因表达的卡介苗胞壁活性蛋白 ,开发增强粘膜抗菌肽基因表达的免疫增强剂为防治粘膜感染的研究打基础。方法 采用超声破碎细胞、蔗糖密度梯度离心、SephadexG 15 0柱层析等方法分离卡介苗胞壁蛋白质组分 ;应用逆转录聚合酶链反应 (RT PCR)法和Northern杂交检测SPC A 1细胞hBD 1mRNA的表达 ;采用琼脂糖弥散杀菌法检测SPC A 1细胞培养上清的抗菌活性。结果 利用RT PCR和Northern杂交分析 ,证实卡介苗刺激SPC A 1细胞hBD 1mRNA的表达增强了 2倍。卡介苗胞壁蛋白SephadexG 15 0层析所得 6个组分中 ,组分 5 [相对分子质量 (Mr)范围在 2 1× 10 3～ 2 9× 10 3]诱导SPC A 1细胞hBD 1mRNA的表达增强 4倍 ,其培养上清的抗菌活性显著增强。这种诱导作用具剂量和时间依赖关系。结论 卡介苗能增强人肺腺上皮SPC A 1细胞hBD 1mRNA的表达及其抗菌活性 ;Mr 为 2 1× 10 3～ 2 9×10 3
Objective To isolate and identify the bioactive proteins that stimulate hBD 1 gene expression in human pulmonary gland epithelial cells, so as to develop a new antibacterial strategy that enhances mucosa antibiotic peptide expression for prevention and treatment of mucosal infections. Methods BCG cells were broken by supersonic technique. The cell wall proteins were isolated by sucrose density gradient centrifugation, and fractionated by Sephadex G 150 column chromatography. Using reverse transcription PCR (RT PCR) and Northern blot analysis, hBD 1 mRNA expression in SPC A 1 cells was examined. The antibacterial activity of the cell culture supernatant was determined by agarose radial diffusion assay. Results Heat inactivated BCG induced two fold enhancement of hBD 1 mRNA expression in SPC A 1. Among 6 fractions of BCG cell wall proteins, only fraction 5 showed an activity that remarkably enhanced hBD 1 mRNA expression in SPC A 1 cells and augmented antibacterial activity of the cell culture supernatant. SDS PAGE showed its M r of 21×10 3 to 29×10 3. The enhanced expression of hBD 1 mRNA was dose and time dependent. Conclusions These results provided for the first time that M. Bovis could stimulate hBD 1 mRNA expression and enhance antibacterial activity of epithelial SPC A 1 cells in human pulmonary gland. Sephadex G 150 fraction 5 of its cell wall proteins (21×10 3 to 29×10 3) was responsible for this effect.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2001年第4期400-403,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目 (39970 2 93
396 70 30 5 )
CMB(98 6 81)
