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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321 bp的AP1和660 bp的BP主要抗原域,经重叠延伸PCR获得966 bp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45 ku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.“,”Objective To construct a recombinant vector expressing the main antigenic domains of the ancillary protein AP1 and backbone pilin (BP) of Streptococcus agalactiae.The expressed product was identified and its antigenicity was analyzed.Methods Protean DNAstar was used to screen for the major antigenic domains of AP1 and BP,and 15 flexible peptides were introduced in order to ensure that the antigens were accessible.The AP1 and BP genes were fused using overlap extension PCR.The gene fragment was amplified,cloned into a pET-30a(+) vector,and transformed into BL21 (DM3) cells for efficient protein expression.The target protein was purified using affinity chromatography and identified using Western blot analysis.Results Amplification with PCR indicated that the major antigen domain of AP1 was 321 bp in length while the major antigen domain of BP was 660 bp in length.Overlap extension PCR yielded a tandem gene fragment of AP1 and BP that was 966 bp in length.DNA sequencing indicated that there were no base deletions,mutations,or shifts.A recombinant expression vector was constructed and protein expression was induced with IPTG to solubly express the target protein,which was about 45 ku.The purity of the purified recombinant protein was above 95 ％.Western blot analysis indicated that the recombinant protein was recognized by serum containing Streptococcus.Conclusion Major antigenic domains of AP1 and BP were obtained using overlap extension PCR.The recombinant expression vector PET-30a(+)/AP1+BP was constructed to induce expression of the target protein,and the product has good immune reactivity.This study has provided an experimental basis for further research on vaccines based on AP1 and BP proteins