狂犬病病毒BD-06株基质蛋白的原核表达、纯化及其多克隆抗体的制备

Prokaryotic Expression and Purification of Matrix Protein of BD-06 Strain Rabies Virus and Preparation of its Polyclonal Antibody

本研究旨在获得抗狂犬病病毒M蛋白的多克隆抗体,为进一步研究M蛋白功能提供材料。本试验以狂犬病病毒BD06毒株为模板克隆M基因序列,将其克隆至原核表达载体pET28a;经酶切和测序鉴定,M蛋白基因序列已正确插入表达载体pET28a;以表达载体pET28a-M转化E.coli Rosetta株,并以0.5mmol/L IPTG诱导,结果表达出27ku左右的M蛋白;将诱导表达的蛋白质回收纯化,以纯化的M蛋白多次免疫兔子制备多克隆抗体;Western blotting分析和间接免疫荧光分析结果表明,制得的抗体与病毒能够反应,运用制得的多克隆抗体定位了基质蛋白在感染狂犬病病毒的BHK-21细胞中的位置。结果表明成功制得了抗狂犬病病毒M蛋白的多克隆抗体,为研究狂犬病病毒M蛋白功能奠定了基础。

The objective of this study was to obtain polyclonal antibody against the matrix protein of rabies virus in order to provide materials for further study on the function of matrix protein. The full sequence of the matrix protein gene from the BD-06 strain rabies virus was successfully cloned and inserted into the expression vector pET28a. The results of restriction enzyme digesting and sequencing proved that the matrix protein gene was correctly inserted into pET28a. After the expression plasmid was transformed into E.coli Rosetta and induced by 0.5 mmol/L IPTG, 27 ku M protein was obtained. After the matrix protein was purified and vaccinated the rabbit,the polyclonal antibody was obtained. The results of Western blotting and IFA showed that the antibody could react with the rabies virus. The matrix protein in the BHK-21 cell affected by BD-06 strain rabies virus was positioned by using the polyclonal antibody. The results indicated that the polyclonal antibody against the matrix protein of rabies virus was successfully prepared, which laid a foundation for further study on matrix protein of rabies virus.