伽氏疏螺旋体尚志株P66基因的原核表达与鉴定

摘要

为获得伽氏疏螺旋体尚志株（B ．garinii SZ）的 P66蛋白，本研究从培养的螺旋体菌液中提取总 RNA，反转录合成第一链 cDNA，利用 PCR 扩增 P 66基因。将目的片段连接表达载体 pET-30a（＋），并转化大肠杆菌表达菌 BL21（DE3），经 PCR、双酶切及测序验证正确后，进行 IPTG 诱导表达和纯化，然后将纯化的融合蛋白免疫新西兰大白兔，制备多克隆抗体。SDS-PAGE 结果显示，获得约70 ku 的表达产物；Western blotting 分析表明，表达产物与抗 His 标签的小鼠单克隆抗体、螺旋体鼠源阳性血清、兔抗 P66多克隆抗体均能发生反应，获得的多克隆抗体也可识别天然蛋白。本试验成功表达了 B ．garinii SZ 株 P66蛋白，并制备了多克隆抗体，为后续 B ．garinii SZ 株P66蛋白的功能研究奠定了基础。%In order to obtain the P66 protein from Borrelia garinii (B.garinii)SZ,the first strand cDNA was synthesized based on the total RNA extracted from B.garinii SZ,and then the targeted P66 gene was amplified by PCR.The fragment was linked into the pET-30a(+)vector,and transformed in-to Escherichia coli BL21(DE3).After identified by PCR,double restriction enzyme digestion,and nu-cleotide sequencing,the recombinant protein was expressed and purified.Polyclonal antibody was then prepared from New Zealand rabbit immunized with purified recombinant protein.The recombinant pro-tein was about 70 ku in size confirmed by SDS-PAGE,and Western blotting analysis indicated that the recombinant P66 protein could recognize the mouse monoclonal anti-His-tag,positive sera of spirochete from mouse,and anti-P66 polyclonal antibody.Additionally,the anti-P66 polyclonal antibody could rec-ognize native P66 protein.In this study,we successfully expressed the recombinant P66 protein and ob-tained the anti-P66 polyclonal antibody,which provided the foundation for further functional studies of P66 protein from B.garinii SZ.