猪丹毒杆菌SpaA基因的克隆与生物信息学分析

Cloning,sequecing,secondary structure and B-cell epitopes prediction of swine Erysipelothrix rhusiopathiae SpaA

旨在克隆猪丹毒杆菌表面保护性抗原A(surface protein A,SpaA)的编码基因,并预测该蛋白的二级结构和B细胞抗原表位,从而探讨猪丹毒杆菌SpaA蛋白在免疫保护中所起的作用。首先对猪丹毒杆菌SpaA基因进行PCR扩增、克隆和测序,并进行同源性比较。应用生物信息学方法,对猪丹毒杆菌SpaA蛋白的二级结构和B细胞抗原表位进行预测。结果显示,猪丹毒杆菌SpaA基因全长1 881bp,编码626个氨基酸,发育进化树结果表明分离的2株猪丹毒杆菌菌株与GenBank中其他菌株在进化树中关系较远,自成一个分支。二级结构的预测结果显示该蛋白主要以无规则卷曲、α螺旋和延伸链为主,只有少数的β转角;推测该蛋白有14个B细胞优势抗原表位区域、8个N-肉豆蔻酰化位点。该研究为进一步分析猪丹毒杆菌免疫机理、表位疫苗设计等奠定理论基础。

The aims of this study was to clone and sequence the SpaA gene of Erysipelothrix rhu-siopathiae and predict its secondary structure and B-cell epitopes, which provides a theoretical ba-sis for the study of immune mechanism and subunit vaccine. The SpaA gene was amplified,cloned, sequenced and phylogenetic analyzed. By using bioinformatics methods, the secondary structure and B-cell preponderant epitope of SpaA were predicted. The results showed that the spaA gene is 1 881 bp,encoding 626 amino acids. The phylogenetic analysis of the SpaA gene indicated the dis-tance between 2 separated strain of Erysipelothrix rhusiopathiae and those of GenBank was far, and 2 separated strain became a new branch. Using the SOPMA method,prediction of the seconda-ry structure of the SpaA showed thata-helix, random coils and extended strand were the main structural type of the flexible region in secondary structure, and contain a small amount of β-turn. It was supposed the SpaA protein contain 14 potential antigen epitopes and 8 potential N-myristoylation sites. These results provided a theoretical basis for the further study of immune mechanisms and epitope vaccine design for the pathogen.