GII.6型诺如病毒P蛋白原核表达及多克隆抗体制备

Prokaryotic expression of GII.6 norovirus P protein and preparation of polyclonal antibody

目的原核表达GII.6型诺如病毒(norovirus,NoV)P蛋白和制备多克隆抗体。方法扩增NoV GII.6型P区基因并克隆至pET28a载体,转化感受态E.coli BL21(DE3),IPTG诱导进行原核表达。使用Ni-NTA亲和柱层析进行纯化,重组蛋白进行寡糖结合分析,免疫BALB/c小鼠制备多克隆抗体血清,ELISA测定该抗体的效价,western blot(WB)检测抗体的特异性,并进行有效性评估。结果成功构建了原核表达载体GII.6P-pET28a并表达相对分子质量约40×10^3的GII.6型P蛋白;Ni柱亲和层析纯化后纯度达90%以上;寡糖结合显示GII.6型P蛋白与B、leb、H2型结合,与A、H1、lea型不结合;ELISA测得抗体的效价为1∶160000;WB显示抗体具有较高特异性;交叉实验未显示出对GII.4的亲和力。结论成功表达了GII.6型P蛋白并获得高效价的抗体,为GII.6的检测和疫苗研发提供有效工具。

Objective To get norovirus(NoV)GII.6 P protein through prokaryotic expression and prepare the polyclonal antibody.Methods NoV GII.6 P region gene was amplified and cloned into prokaryotic expression vector pET28a,and the recombinant plasmid was transformed into E.coli BL21(DE3)competent cell.The recombinant protein GII.6 P was expressed by induced Isopropylβ-D-Thiogalactoside(IPTG)and then purified with Ni-NTA Affinity Column.The binding ability of recombinant GII.6 P was determined by oligosaccharide binding assay and the polyclonal antibody serum was prepared by immunizing BALB/c mice.The titer of GII.6 P polyclonal antibody was determined by ELISA,and the specificity of the antibody was detected by western blot(WB).The effectiveness of GII.6 P polyclonal antibody was assessed.Results The recombinant GII.6P-pET28a plasmid was constructed successfully and the recombinant GII.6 P protein was expressed with relative molecular mass of 40×10^3.The purity of GII.6P protein was more than 90%after purification.The oligosaccharide binding showed that the GII.6 P protein binds to B,leb and H2,but does not bind to A,H1,and lea type;the titer of GII.6 P polyclonal antibody was 1∶160000.WB indicated that the antibody had high specificity and the cross experiments did not show affinity to GII.4.Conclusions The GII.6P protein has been expressed successfully and the GII.6 P polyclonal antibody with high titer was prepared,which provides an effective tool for detection and vaccine development for NoV GII.6.