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

发布时间：2018-06-17 02:53

本文选题：无乳链球菌 + sip基因　；参考：《中国兽医学报》2017年07期

【摘要】：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。
[Abstract]:To obtain multiple subunit fusion proteins with triple activity of streptococcus lactococcus membrane associated protein (sip), phosphoglycerin kinase (pgk) and fibronectin FbsA (FbsA), the feasibility of using them as candidate antigens of streptococcus lactococcus vaccine was studied. According to the nucleotide sequences of Sippgk and FbsA genes of Streptococcus lactis reported in GenBank, the antigen epitopes of the protein were analyzed by DNA Star software and 4 pairs of primers including overlapping PCR primers were designed and synthesized. The first two major antigen regions were spliced and inserted into pET30a () vector by overlapping PCR technique, and the third gene was inserted into pET30a () vector. In order to reduce the interference of spatial conformation between the three proteins in the process of triple gene tandem expression, the flexible linker of 45bp was introduced into each of the three gene junctions, and the recombinant gene was expressed soluble in BL21 receptive state. Western blot analysis showed that the fusion protein could be recognized by streptococcus lactobacillus polyantibodies, and the fusion protein could recognize three proteins such as sip pgk and FbsA. These results suggested that the constructed fusion protein may have triple activity of sip pgk and FbsA proteins, and the recombinant polysubunit fusion antigen was superior to a single protein in protecting mice against toxin attack. This study provides some reference data for the study of new vaccine of bovine streptococcal mastitis.
【作者单位】：内蒙古民族大学生命科学学院;内蒙古民族大学乳源性致病菌研究所;山东省滨州市畜牧兽医研究所;
【基金】：国家自然科学基金资助项目(31060352,31260600) 内蒙古民族大学与通辽市政府合作资助项目(SXYB2012086)
【分类号】：S858.23

【参考文献】