金黄色葡萄球菌表面蛋白的表达及其多克隆抗体的制备

Expression of Staphylococcus aureus surface protein and preparation of its polyclonal antibody

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 the fibronectin-binding protein A（FnBPA） of Staphylococcus aureus in prokaryotic cells and prepare its polyclonal antibody. Methods The extracellular fragment with antigen epitopes of FnBPA was selected,of which the gene sequence was optimized and cloned into vectors pGEX4T-2 and pET-28a（＋）respectively. The constructed recombinant plasmids pGEX-4T-2-fnbpa and pET-28a-fnbpa were transformed to E. coli BL21（DE3） for expression under induction of IPTG. The fusion proteins FnBPA-GST and FnBPA-His were purified by High Affinity GSTNTA resion and High Affinity Ni-NTA resin. Polyclonal antibody was prepared by immunizing New Zealand white rabbits with the purified FnBPA-His fusion protein,determined for titer by indirect ELISA using FnBPA-GST fusion protein as detection antigen,and for specificity by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmids pGEX4T-2-fnbpa and pET-28a-fnbpa were constructed correctly. Fusion proteins FnBPA-GST and FnBPA-His,with relative molecular masses of about 42 000 and 19 000,contained about 30% and 20% of total somatic proteins,respectively. Both the fusion proteins were expressed in soluble forms,and reached purities of more than 90%.High titer antiserum was obtained by immunizing New Zealand white rabbits. Conclusion Both the expressed fusion proteins showed high immunogenicity and antigenicity,and the prepared polyclonal antibody showed high specificity,which laid a foundation of development of detection method for Staphylococcus aureus.