人星状病毒1型衣壳蛋白原核表达、纯化及多克隆抗体制备的研究

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。

Molecular cloning and expression of human astrovirus serotype 1 capsid protein and preparation of the respective polyclonal antibodies

Objective To express the VP26 fragment of human astrovirus serotype 1(HAstV-1) capsid protein in prokaryotic system,generate the respective polyclonal antibodies and establish the antibody sandwich enzyme-linked immunosorbent assay(ELISA) for detecting the protein,so as to lay the foundation for larger scale expression of VP26 protein,generation of genetic engineered vaccine and quick detection of the disease in clinic.Methods The recombinant VP26(rVP26) protein was cloned and expressed in Escherichia coli(E.coli),and purified by Ni2+-NTA agarose affinity chromatography.Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE),bicinchoninic acid(BCA) assay and Western blot(WB).The serum of rabbits immunized with rVP26 was collected from heart as the polyclonal antibody.The titer and specificity of rabbit antiserum was measured by ELISA.Results The prokaryotic expression vector pET30a(+)-VP26 was successfully constructed and the rVP26 protein was highly expressed in E.coli(Rosetta 2) cells.The titer of antiserum measured by ELISA was 1 ∶ 8 000,which satisfies the experimental requirements.Conclusion It is demonstrated that the establishment of prokaryotic expression system and preparation of the polyclonal antibodies could provide the foundation for the future studies.They would be helpful for further studying the pathogenesis of HAstV-1 infection and development of HAstV-1 vaccines as the immunological tools.