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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-BcAg1~214aa)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。
Objective: To construct a prokaryotic expression plasmid of truncated duck hepatitis B virus (DHBV) core protein and express it in E. coli to prepare polyclonal antibodies. Methods: The gene fragment encoding the truncated DHBV core protein (DH-BcAg1 ~ 214aa) was inserted into the prokaryotic expression vector pRSET-B and induced in host Rosetta (DE3) pLacI by gene engineering. NTA method to purify the target protein. The purified recombinant protein was used to immunize BALB / c mice to prepare polyclonal antibody. The sensitivity and specificity of the antibody were tested by enzyme - linked immunosorbent assay (ELISA), Western blot and immunohistochemistry. Results: The plasmid containing the truncated DHBV core region was successfully constructed and the target protein with a relative molecular mass (Mr) of about 28000 was purified. Immunization of BALB / c mice resulted in high specific titer Clone antibody. CONCLUSION: The obtained recombinant truncated DHBV core antigen has high purity and strong immunoreactivity. The obtained polyclonal antibody has high potency and good specificity, which lays the foundation for the detection and research of DHBV.