金黄色葡萄球菌表面蛋白SdrD的原核表达及抗体制备

摘要

根据金黄色葡萄球菌表面蛋白SdrD基因序列设计特异性引物，以金黄色葡萄球菌标准菌株基因组为模板进行PCR扩增，获得了大小为2043 bp的基因片段。将基因片段连接至表达载体 pET26b，转入大肠埃希菌BL21（DE3）中用IPTG诱导表达。SDS-PAGE结果表明，SdrD基因获得了较好的表达，重组蛋白的分子质量约为99 ku。利用免疫亲和层析对目标蛋白进行纯化，获得了纯度较高的目标蛋白。用该蛋白免疫新西兰白兔制备了多克隆抗体，抗体效价达到1∶23000，为金黄色葡萄球菌基因工程疫苗的开发与性能评价，以及该致病菌免疫检测技术的开发奠定了基础。%In this study,specific primers were designed and synthesized based on the SdrD gene of Staphy-lococcus aureus and used to amplify the gene fragment using the genomic DNA of S.aureus standard strain as template.A gene fragment of 2 043 bp in length was obtained.The ORF was ligated into pET26b ex-pression vector and transformed into E.coli BL2 1 (DE3 )for recombinant expression under induction of IPTG.The results of SDS-PAGE indicated that the recombinant protein with molecular weight of 9 9 ku was successfully expressed.The target protein was purified using immunoaffinity chromatography and used to immunize New Zealand white rabbits to prepare polyclonal antibodies.ELISA demonstrated that the titers of antiserum reached 1∶23 000.This study lays a foundation for developing engineered vaccines and immune detection methods for S.aureus.