马链球菌兽疫亚种串联表达蛋白对小鼠免疫效果的评价

Immunity effect assessment in mice of a tandem fusion protein composed of M-like protein,fibronectin-binding protein and IgG-binding protein of Streptococcus equi ssp.zooepidemicus

根据马链球菌兽疫亚种类M蛋白（SzP）、纤维结合素结合蛋白（FNZ）、IgG结合蛋白（ZAG）的基因序列,分别设计并合成引物。以马链球菌兽疫亚种ATCC35246的基因组为模板,扩增szp（527-1 044bp）、fnz（1 012-1 519bp）、zag（134-664bp）基因序列,并构建原核表达载体pET-32a（＋）-szp、pET-32a（＋）-fnz、pET-32a（＋）-zag。确定诱导表达的三种蛋白都具有免疫原性后,通过PCR串联的方法,将以上3个目的片段同时串联克隆到表达载体pET-32a（＋）中。重组质粒转化入大肠杆菌BL21后,经IPTG诱导表达分子质量约为74ku的串联融合蛋白SzP-FNZ-ZAG。免疫印迹分析结果表明,上述融合蛋白能够与ATCC35246鼠源多克隆抗体发生结合反应。分别用纯化的单个蛋白和串联重组蛋白免疫ICR小鼠,能够诱导免疫小鼠产生较高效价的血清抗体,串联重组蛋白对致死剂量的马链球菌兽疫亚种强毒菌株的攻击具有90%的免疫保护率,显著高于单个蛋白。RT-PCR检测显示,免疫小鼠IL-4、IFN-γ基因的转录水平显著高于空白对照组小鼠。

Three fragments of szpgene（527-1 044bp）,fnz gene（1 012-1 519bp）and zaggene（134-664bp）were amplified from genomic DNA of Streptococcusequissp.zooepidemicus（SEZ）by PCR,and the recombinant expression vectors were constructed respectively.After the immunogenicity of the three recombinant proteins was confirmed,the three genes were all cloned into the plasmid vector pET-32a（＋）via restriction endonucleases NcoⅠ and XhoⅠ,and then transformed into host strain BL21.The 74 ku fusion protein was expressed in BL21 after being induced with IPTG.Western-blot analysis was carried out by using the antiserum of ATCC35246.The results of Western-blot showed that the fusion protein SzPFNZ-ZAG had binding capacity with polyclonal antibody of ATCC35246.To compare the immune protective efficacy,ICR mice were immunized with the single and tandem recombinant proteins（SzP,FNZ,ZAG and SzP-FNZ-ZAG）,respectively.The immune protection of the tandem recombinant protein SzP-FNZZAG was 90%,which was obviously higher than the other three single recombinant proteins.RT-PCR analysis showed a significantly increase in the level of IL-4and IFN-γ mRNA in the immunized mice.