猪链球菌2型MRP与EPF基因串联表达蛋白及其免疫保护作用

Cloning and Expression of a Fusion Protein of Muramidase-Released Protein and Extracellular Protein Factor of Streptococcus suis Type 2 and Its Protection

根据猪链球菌2型(Streptococcus suis type 2,SS2)溶菌酶释放蛋白(muramidase-released protein,MRP)和胞外蛋白因子(extracellular protein factor,EPF)的基因序列,各设计合成一对引物,以猪链球菌2型江苏分离株SS2-1的基因组为模板,扩增mrp基因1 801~2 513位序列和epf 基因1 783~2 563位序列,分别构建原核表达载体pET32a-mrp 、pET32a-epf,确定诱导表达的两种蛋白都具有免疫原性后,提取阳性克隆质粒各自进行双酶切并纯化,通过PCR串联两片段,将目的片段定向克隆到表达载体pET-32a中,重组质粒转化入大肠杆菌BL21,经IPTG诱导表达分子量约为74kD的融合蛋白.用制备的两种抗血清与纯化融合蛋白进行免疫转印,结果显示融合蛋白分别具有MRP与EPF的中和表位.用融合蛋白MRP-EPF免疫新西兰兔,以最小致死量猪链球菌强毒株SS2-1攻击,兔的存活率达50%(2/4),存活率明显高于单个表达产物.证实串联表达的融合蛋白为重要的保护性抗原.

Two fragments of mrp gene (1 801-2 513bp) and epf gene (1 783-2 563bp ) were amplified from genomic DNA of Streptococcus suis type 2 isolate strain SS2-1 by polymerase chain reaction (PCR) technique. The PCR products were later cloned into plasmid vector pET-32a via restriction endonuclease and then transformed into host strain BL21. Through relevant endonuclease digest , the positive recombinant bacteria were identified, which were expressed by IPTG of optimal concentration, respectively. Also, the antigenicity of expressed recombinant proteins (rMRP and rEPF) were determined by Western-blot. After verified positive immunity, the fragments of mrp and epf linked by PCR was cloned into pET-32a again. By the above method with IPTG, the recombinant protein consisted of rMRP and rEPF with molecular weight 74kD was obtained. Next, the antigenicity analysis by Western-blot showed that the recombinant protein had the conserved epitopes of MRP and EPF, respectively. In order to further determine the antigenicity of recombinant proteins (rMRP, rEPF and rMRP-EPF), rabbits were immunized, positive (immunity with bacterial vaccine) and negative controls (non-immunity) were set. After 21d, challenged by Streptococcus suis type 2 isolate strain SS2-1,rMRP-EPF immunized rabbits were protected more than alone. Compared to rMRP 25%,rEPF 0%, bacterial vaccine 100%, rMRP-EPF beared protection rates of 50%. It is concluded that rMRP-EPF possesses good antigenicity.