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目的表达纯化人星状病毒1型(HAstV-1)衣壳蛋白VP26片段,制备多克隆抗体,初步建立夹心酶联免疫吸附分析(ELISA)检测病毒抗原方法,为进一步大量表达VP26片段、构建基因工程疫苗和临床检测奠定基础。方法利用原核表达系统在大肠杆菌中克隆、表达重组VP26蛋白,并以Ni2+-NTA亲和层析法纯化重组蛋白,运用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、二噻啉甲酸法(BCA)实验、Western blot等方法对重组蛋白纯度、浓度、抗原性进行评价鉴定。以重组VP26蛋白作为抗原,免疫新西兰大耳兔获得多抗血清并对其效价、特异性用ELISA鉴定。结果原核表达载体pET30a(+)-VP26构建成功,重组VP26蛋白可在大肠杆菌Rosetta 2宿主菌中大量表达。免疫兔所得多抗血清效价达到1∶8 000,可以满足夹心法ELISA实验的需要。结论HAstV-1衣壳蛋白原核表达系统建立和多克隆抗体制备可以作为今后相关研究的基础,有助于对HAstV-1感染致病机制、免疫诊断和疫苗研制的更深入研究。
Objective To express and purify human capsid protein VP26 fragment of human astrovirus type 1 (HAstV-1), and to prepare polyclonal antibody. The method of sandwich enzyme-linked immunosorbent assay (ELISA) to detect viral antigen was established. Engineering vaccines and clinical testing to lay the foundation. Methods The recombinant VP26 protein was cloned and expressed in Escherichia coli using prokaryotic expression system. The recombinant protein was purified by Ni2 + -NTA affinity chromatography. The recombinant protein was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) , Dithianic acid method (BCA) assay, Western blot and other methods to evaluate the purity, concentration and antigenicity of the recombinant protein. Recombinant VP26 protein was used as antigen to immunize New Zealand big-eared rabbits to obtain multi-antiserum and its titer was determined by ELISA. Results The prokaryotic expression vector pET30a (+) - VP26 was successfully constructed. The recombinant VP26 protein was highly expressed in E. coli Rosetta 2. The antiserum titer obtained by immune rabbits reached 1: 8000, which can meet the needs of sandwich ELISA experiments. Conclusion The establishment of prokaryotic expression system of HAstV-1 capsid protein and the preparation of polyclonal antibody could serve as the basis for future research and help to further study the pathogenesis, immunological diagnosis and vaccine development of HAstV-1 infection.