牛乳腺炎无乳链球菌PI-2a菌毛岛辅助蛋白AP1、BP主要抗原域串联表达及其免疫活性鉴定

发布时间：2018-04-25 16:51

  本文选题：无乳链球菌 + 菌毛岛屿　； 参考：《中国病原生物学杂志》2017年07期

【摘要】：目的构建无乳链球菌菌毛岛屿辅助蛋白AP1、菌毛骨架蛋白BP主要抗原域串联表达重组载体,对表达产物进行抗原性分析及鉴定。方法利用DNAstar Protean功能筛选AP1和BP主要抗原结构域,引入15位柔性多肽,通过重叠延伸PCR技术串联融合AP1、BP主要抗原决定簇基因区,PCR扩增串联体基因片段,克隆至pET-30a(+)载体后转化BL21(DE3)细胞,表达的目的蛋白经亲和层析纯化后进行Western blot鉴定。结果 PCR扩增出321bp的AP1和660bp的BP主要抗原域,经重叠延伸PCR获得966bp的AP1、BP串联体基因片段,DNA测序表明无碱基缺失、突变和移码。构建的重组表达载体经IPTG诱导能可溶性表达分子质量约45ku的目的蛋白,纯化后重组蛋白的纯度≥95%,Western blot分析表明,重组融合蛋白能被兔源抗无乳链球菌阳性血清识别。结论重叠延伸PCR获得AP1、BP主要抗原域串联体,构建的pET-30a(+)/AP1+BP重组表达载体能以包涵体形式表达目的蛋白,表达产物具有良好的反应原性,为研究基于AP1、BP蛋白的致病机制及其免疫活性奠定了实验基础。
[Abstract]:Objective to construct the recombinant vector expressing the main antigen domain of Streptococcus lactis tuber island helper protein AP1 and its skeleton protein BP in tandem, and to analyze and identify the antigenicity of the expressed product. Methods the main antigenic domains of AP1 and BP were screened by DNAstar Protean function, and 15 position flexible polypeptides were introduced. The tandem gene fragments were amplified by tandem fusion of AP1GBP major antigen-determinant gene region by overlapping extension PCR technique. The target protein was cloned into pET-30a () vector and transformed into BL21DE-3 cells. The expressed protein was purified by affinity chromatography and identified by Western blot. Results the main antigenic domains of 321bp AP1 and 660bp BP were amplified by PCR. The sequence of 966bp AP1 BP tandem gene fragment obtained by overlapping extension PCR showed that there was no base deletion, mutation and frameshift. The recombinant expression vector was induced by IPTG to express the target protein with molecular weight of about 45ku. The purity of purified recombinant protein was 鈮，

本文编号：1802164