摘要
目的：构建截短型鸭乙型肝炎病毒（DHBV）核心蛋白的原核表达质粒并在大肠杆菌中表达，制备多克隆抗体。方法：应用基因工程技术将编码截短型DHBV核心蛋白（DH-BcAg 1～214aa）的基因片段装入原核表达载体pRSET-B内，在宿主菌Rosetta（DE3）pLacI内进行诱导表达，运用Ni-NTA方法纯化目的蛋白。用纯化的重组蛋白免疫BALB／c小鼠制备多克隆抗体，并采用酶联免疫吸附实验（ELISA），Western blot及免疫组化检测抗体的灵敏度和特异性。结果：成功地构建了含截短型DHBV核心区基因的质粒，并纯化得到了相对分子质量（Mr）约为28000的目的蛋白，用之免疫BALB／c小鼠获得了高效价的特异性多克隆抗体。结论：获得的重组截短型DHBV核心抗原纯度高，免疫反应性强；获得的多克隆抗体有较高的效价和较好的特异性，为DHBV的检测和研究奠定了实验基础。
AIM： To construct a prokaryotic plasmid expressing truncated duck hepatitis B virus core protein （ DHBc1～214）, purify the recombinant protein, and to develop polyclonal antibodies against DHBc. METHODS： DHBc1～214 was cloned into vector pRSET-B, then expressed in E. coli Rosetta （DE3） pLacl induced by IPTG. The recombinant protein was purified using Ni-NTA spin column. Polyclonal antibody was developed by immunizing BALB/c mice with the purified recombinant protein, and their sensitivity and specificity were tested using enzyme-linked immunosorbent assay, immunohistochemical staining and Western blot analysis. RESULTS： Recombinant plasmid expressing truncated DHBc1～214 was successfully constructed. A protein of 28 000 was expressed and purified. Polyclonal serum antibody with a high specificity was obtained by immunizing BALB/c mice with the purified recombinant protein. CONCLUSION： The truncated recombinant DHBc1～214developed in this study is purified and shown strong antigenecity. The polyclonal antibody against DHBc protein is generated by regular immunization method, demonstrating both high sensitivity and specificity. The protein and the antibody can be used for further clinical examination and research of DHBV.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2008年第5期467-470,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30571646)
关键词
鸭乙型肝炎病毒
核心蛋白
原核表达载体
多克隆抗体
duck hepatitis B virus
core protein
prokaryotic expression vector
polyclonal antibody
