巴什拜羊重组BPI蛋白的体外功能检测
Function of Recombinant BPI Protein of Bashiby Sheep in Vitro

研究巴什拜羊重组杀菌性/通透性增加蛋白（recombinant bectericidal/permeability increasing protein，rBPI）的体外生物学特性。向体外培养的鼠脑内皮细胞MBECs（mouse brain endothelial cell）中加入rBPI，利用Annexin V FITC/PI染色法在荧光显微镜下鉴定凋亡细胞，再用琼脂糖凝胶电泳观察DNA ladder，以检测rBPI诱导MBECs凋亡的作用；在体外培养的绵羊肺炎支原体（Mycoplasma ovipneumoniae，MO）中加入rBPI，利用real-time PCR技术检测rBPI对MO的抑制作用；在体外培养的大肠杆菌中加入rBPI，利用微量肉汤稀释法检测rBPI的抑菌作用；在体外培养的绵羊中性粒细胞中加入MO，利用real-time PCR技术检测BPI mRNA的表达水平。结果表明，Annexin V FITC/PI染色法可观察到凋亡早期细胞呈绿色，凋亡晚期或死亡细胞呈红色，在540 nmol/L rBPI浓度诱导作用下可见清晰的DNA ladder条带；rBPI作用于MO 3 h 时，MO16S rRNA表达水平显著低于对照组（P＜0.05）；20、40和80 mg/L的rBPI与阳性对照组相比均能够显著抑制大肠杆菌的生长(P＜0.05)；绵羊中性粒细胞受到MO感染后3 h和4 h时表达BPI mRNA的水平均显著高于对照组(P＜0.05)。
To study the biological characteristics of rBPI (recombinant bectericidal/permeability increasing protein，rBPI) in vitro.After adding rBPI to MBECs (mouse brain endothelial cells) cultured in vitro,the method of Annexin V FITC/PI staining were used to identify apoptotic cells under fluorescence microscope,and then to detect DNA ladder by agarose gel electrophoresis;adding rBPI to MO(Mycoplasma ovipneumoniae，MO) to detect the inhibition of rBPI on MO by real-time PCR;method of broth microdilution antifungal susceptibility was used to detect the inhibition of rBPI on E.coli; adding MO to the Bashiby sheep neutrophils isolated and cultured in vitro to detect BPI mRNA expression level of neutrophils by real-time PCR.The results showed that the early apoptotic cells were green and the late apoptotic or dead cells were red while it was observed by the method of Annexin V FITC/PI staining,and DNA ladders of MBECs were clearly detectable at 540 nmol/L of rBPI；the 16S rRNA expression level of MO which had been treated by rBPI 3 h was significantly lower than that of the control group(P＜0.05); 20,40 and 80 mg/L of rBPI can significantly inhibit E.coli than the control group(P＜0.05);the BPI mRNA expression level of neutrophils which had been treated by MO 3 h and 4 h were higher than that of control group (P＜0.05).