哺乳动物正呼肠孤病毒血清3型Dearingσ1蛋白纯化及其多克隆抗体制备

Purification of mammalian orthoreovirus serotype 3 Dearingσ1 protein and preparation of polyclonal antibody

目的纯化哺乳动物正呼肠孤病毒(MRV)血清3型Dearing(T3D)σ1融合蛋白,制备T3Dσ1多克隆抗体。方法将T3DS1-pET28a重组质粒转化至Rosetta(DE3)感受态细胞,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导目的蛋白大量表达,经组氨酸标签Ni-IDA层析柱纯化得到T3Dσ1融合蛋白。纯化得到的蛋白免疫新西兰白兔制备σ1蛋白特异性多克隆抗体。通过间接ELISA检测多克隆抗体效价,Western blot法及间接免疫荧光法(IFA)检测多克隆抗体特异性。结果T3Dσ1蛋白相对分子质量(Mr)约为32000,主要为包涵体形式,通过变性、复性处理纯化融合蛋白;免疫新西兰白兔成功制备出T3Dσ1效价大于1∶10^(6)的多克隆抗体,并成功应用于Western blot法及IFA检测。结论成功制备了高效价及高灵敏度T3Dσ1多克隆抗体。

Objective To prepare T3Dσ1 polyclonal antibody with purified σ1 fusion protein of mammalian orthoreovirus(MRV) serotype 3 Dearing(T3D). Methods The recombinant plasmid T3DS1-pET28a was transformed into Rosetta(DE3) competent cells, and the isopropyl-β-D-thiogalactopyranoside(IPTG) was used to induce a large amount of target proteins which were subjected to purification by histidine-tagged Ni-IDA chromatography column to obtain the T3Dσ1 fusion protein. New Zealand white rabbits were immunized with the purified protein to prepare polyclonal antibodies specific against σ1 protein. The titer of polyclonal antibody was detected by indirect ELISA, and the specificity by Western blot analysis and indirect immunofluorescence assay(IFA). Results The relative molecular mass(M;) of T3Dσ1 fusion protein, mainly in the form of inclusion body, was about 32 000. The fusion protein was purified by denaturation and renaturation. The polyclonal antibody with the titer greater than 1∶10^(6) was prepared by immunizing New Zealand white rabbits and detected by Western blot analysis and IFA. Conclusion The T3Dσ1 polyclonal antibody with high titer and sensitivity was prepared.