奶牛乳腺炎无乳链球菌sip、pgk及FbsA基因主要抗原区域的融合表达及抗原性鉴定

Fusion expression of main antigen area of sip,pgk and FbsA subunit genes and the antigenicity identification in Streptococcus agalactiae for dairy cow mastitis

为获得具有无乳链球菌膜表面相关蛋白sip和磷酸甘油激酶pgk及纤连蛋白FbsA三重活性的多亚单位融合蛋白,研究其作为无乳链球菌亚单位疫苗候选抗原的可行性,根据GenBank中已发表的无乳链球菌sip、pgk及FbsA基因的核苷酸序列,利用DNAStar软件对蛋白的抗原表位序列进行分析并设计合成包括重叠PCR引物共4对引物,通过重叠PCR技术将前2个基因主要抗原区域进行拼接后插入pET30a(+)载体,再将第3个基因插入。为减少三重基因串联表达过程中3个蛋白之间空间构象的干扰,在3个基因连接处各引入1段45bp的柔性linker,结果重组基因在BL21感受态中实现了可溶性表达,表达的蛋白约62 000;Western blot试验表明多亚单位融合蛋白可被无乳链球菌多抗识别,且由此融合蛋白制备小鼠多抗能够识别sip、pgk及FbsA等3个蛋白;这表明构建的多亚单位融合蛋白可能具备sip、pgk及FbsA蛋白的三重活性,而且在小鼠的攻毒保护性试验中,重组多亚单位融合抗原对攻毒小鼠的保护优于单个蛋白。本试验为牛无乳链球菌性乳腺炎新型疫苗的研究提供了一定的参考数据。

In order to get the fusion protein which has the bioactivities of both surface immunogenic protein,phosphoglycerate kinase and fibrinogen binding protein A, and explore the feasibility of the fusion protein as immunoadjuvant. Based on the reported sip,pgk and FbsA gene nucleotide sequences of Streptococcus agalactiae, six primers were designed. The genes sip, pgk and .fbsA were fused by overlapping PCR and inserted into the pETg0a（＋） expression vector. Then the tan- dem gene was inserted into the expression vector, in order to reduce the interference of three pro teins,there was a 45 bp size of linker peptide between the three genes,then the fusion gene was ex- pressed by pET30a （＋） expression vector in BL21 E. toll, a protein can be identified by polyclonal antibody of Streptococcus agalactiae. The mouse polyclonaI antibody prepared by this fusion pro- tein could recognized sip,pgk and FbsA proteins,indicating that the fusion protein has the activity of sip,pgk and FbsA proteins,and the multi subunit recombinant fusion antigens have a excellent protection during the experiment. This study lays a foundation for the development of vaccine for bovine mastitis caused by Streptococcus agalactiae.