伽氏疏螺旋体尚志株P66基因的原核表达与鉴定

发布时间：2018-06-03 15:58

本文选题：伽氏疏螺旋体尚志株 + P　；参考：《中国畜牧兽医》2017年01期

【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P66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70ku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。
[Abstract]:In order to obtain the P66 protein of B. garinii SZ, the total RNAs were extracted from the cultured spirochetes, and the first strand cDNAwas synthesized by reverse transcription. The P66 gene was amplified by PCR. The target fragment was ligated into the expression vector pET-30a (pET-30a) and transformed into E. coli expression strain BL21DDE3. The recombinant protein was induced and purified by IPTG after PCR, double enzyme digestion and sequencing. Then the purified fusion protein was immunized with New Zealand white rabbit. The results of preparation of polyclonal antibody. SDS-PAGE showed that the expressed product of about 70ku could react with mouse monoclonal antibody, spirulina positive serum and rabbit anti-P66 polyclonal antibody. The obtained polyclonal antibodies can also recognize natural proteins. In this experiment, the P66 protein of B.garinii SZ strain was successfully expressed, and the polyclonal antibody was prepared, which laid a foundation for the functional study of P66 protein of B.garinii SZ strain.
【作者单位】：中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室;江苏省动物重要疫病与人兽共患病防控协同创新中心;
【基金】：农业科技创新工程(ASTIP) 国家肉牛牦牛产业技术体系(NBCIS CARS-38)
【分类号】：S852.6

【相似文献】