摘要
目的研究在大肠杆菌中以重组病毒样颗粒形式表达的人II型自身免疫性肝炎标志蛋白CYP2D6主要抗原表位aa262-270免疫小鼠后对构建小鼠自身免疫性肝炎模型的效应。方法设计人II型自身免疫性肝炎标志蛋白主要抗原表位aa262-270(WDPAQPPRD)的正负链寡核苷酸片段,经退火后插入表达乙肝核心蛋白HBcAg的质粒pNP中(pNP质粒由pThioHisA质粒插入HBcAg基因片段构建而成)。构建成质粒pNP-AIH2;转染大肠杆菌DH5α感受态细胞;以IPTG诱导重组蛋白表达,经硫酸铵沉淀、洗涤,密度梯度离心纯化和脱盐后,肌肉注射免疫小鼠3次,以ELISA和western blot检测免疫后小鼠特异性抗体的产生和水平,通过转氨酶检测和肝脏石蜡切片HE染色观察炎症反应。结果 SDS-PAGE结果显示重组菌诱导表达的目的蛋白分子量为19kD,与预期相符;电镜证实其以可溶性的病毒样颗粒形式存在;ELISA显示,免疫小鼠血清与包被的肝脏匀浆蛋白特异反应,检测到持续存在的特异抗体,滴度至少为1:2000;western blot检测表明,抗血清在分子量约为55kD的地方产生了一个特异的条带,与小鼠CYP2D6蛋白大小一致。与正常对照相比,实验组小鼠转氨酶水平未明显升高,肝切片HE染色没有观测到明显的病理特征。结论携带人II型自身免疫性肝炎标志蛋白CYP2D26主要表位的病毒样颗粒免疫小鼠后,可刺激产生较强的特异抗体水平,但在观测时间内,未见诱导小鼠产生明显的肝脏炎症反应。
Objective To evaluate whether recombinant main epitope aa262-270 of marker antigen CYP2D26 in autoimmune hepatitis type II, expressed in E.coli as virus-like particles, will impact on the establishment of mouse autoimmune hepatitis model. Methods Primers ofaa262-270 were designed according to the amino acid sequence WDPAQPPRD, and inserted into plasmid pNP after annealing, pNP was constructed using plasmid pThioHisA expressing hepatitis B core antigen HBcAg. Successfully constructed plasmid was named as pNP-AIH2. The epitope was soluble expressed as virus-like particles in E.coli. After ammonium sulfate precipitation, density gradient centrifuge and desalting, the VLP was used to immune BALB/c mice three times per im. The specific antibody was detected by ELISA and western blot. The inflammation of liver was confirmed by ALT （Alanine aminitransperase） level test and HE staining. Results The peptide existed as soluble virus-like particles with molecular weight of 19.0 kD after expression. The purity of the VLP was proximately 90%. Constant specific antibody was detected in mice serum after immunization with VLP and mean titer was 1：2000. The antibody recognized CYP2D6 protein from mouse liver by western blot specifically forming a band at about 55 kD Elevated ALT level was not detected in immunized mice compared with control group, and no obvious inflammation was observed in liver wax section by HE staining Conclusion Marker antigen CYP2D26 epitope in autoimmune hepatitis type I! expressed in E.coli as virus-like particles elicits elevated antibody level in mice, but can＇t induce obvious liver inflammation during observation
出处
《中国医药生物技术》
2013年第1期24-28,共5页
Chinese Medicinal Biotechnology
基金
云南省应用基础研究面上项目(2010CD135)
重点项目(2011FA023)
