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目的在原核细胞中表达金黄色葡萄球菌纤连蛋白结合蛋白A(fibronectin-binding protein A,FnBPA),制备其多克隆抗体。方法筛选FnBPA抗原表位富集区,优化基因序列,分别克隆至质粒pGEX4T-2和pET-28a(+)中,构建重组表达质粒pGEX4T-2-fnbpa和pET-28a-fnbpa,分别转化大肠埃希菌(E.coli)BL21(DE3),经IPTG诱导表达后,分别用High Affinity GST-NTA resin和High Affinity Ni-NTA resin进行纯化。以纯化后的FnBPA-His融合蛋白为抗原免疫新西兰大白兔,制备多克隆抗体,以FnBPA-GST融合蛋白为检测抗原,采用间接ELISA法检测血清效价,Western blot法检测特异性。结果经双酶切及测序鉴定,证明质粒pGEX4T-2-fnbpa和pET-28a-fnbpa构建正确;在相对分子质量约42 000和19 000处,分别可见FnBPA-GST和FnBPA-His融合蛋白的表达,表达量分别约占菌体总蛋白的30%和20%,均以可溶性形式表达,纯度均在90%以上;免疫新西兰大白兔后获得高效价的特异性兔抗血清。结论2种标签的融合蛋白均具有较好的免疫原性和抗原性,所制备的多克隆抗体具有较好的特异性,为金黄色葡萄球菌检测方法的建立奠定了基础。
Objective To express Staphylococcus aureus fibronectin-binding protein A (FnBPA) in prokaryotic cells and prepare its polyclonal antibody. Methods The enrichment region of FnBPA epitope was screened and the gene sequence was optimized and cloned into pGEX4T-2 and pET-28a (+) respectively. The recombinant plasmids pGEX4T-2-fnbpa and pET-28a-fnbpa were transformed into E.coli E. coli BL21 (DE3) was induced by IPTG and then purified with High Affinity GST-NTA resin and High Affinity Ni-NTA resin respectively. The purified FnBPA-His fusion protein was used as an antigen to immunize New Zealand white rabbits to prepare polyclonal antibody. The FnBPA-GST fusion protein was used as the detection antigen, the indirect ELISA was used to detect the serum titer, and the specificity was detected by Western blot. The results of double enzyme digestion and sequencing showed that the plasmids pGEX4T-2-fnbpa and pET-28a-fnbpa were constructed correctly. The relative molecular mass of about 42 000 and 19 000 at the FnBPA-GST and FnBPA-His fusion protein expression , Which accounted for about 30% and 20% of the total bacterial proteins, respectively. All of them were expressed in soluble form with the purity above 90%. High titer specific rabbit antiserum was obtained after immunization of New Zealand white rabbits. Conclusion The fusion proteins of the two kinds of tag have good immunogenicity and antigenicity. The prepared polyclonal antibodies have good specificity and laid the foundation for the establishment of the detection method of Staphylococcus aureus.