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| 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 | |
| 引用本文： | 李明,何孔旺,陆承平.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[J].中国农业科学,2005,38(6):1264-1269. |
| 作者姓名： | 李明 何孔旺 陆承平 |
| 作者单位： | 1. 南京农业大学/农业部动物疫病诊断与免疫重点开放实验室,南京,210095;江苏省农业科学院兽医研究所/农业部畜禽疫病诊断重点开放实验室,南京,210014 2. 江苏省农业科学院兽医研究所/农业部畜禽疫病诊断重点开放实验室,南京,210014 3. 南京农业大学/农业部动物疫病诊断与免疫重点开放实验室,南京,210095 |
| 基金项目： | 国家“973”项目(G1999011906)，江苏省(三药)科技攻关项目(BE20040620)资助 |
| 摘 要： | 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。 |
| 关 键 词： | 猪链球菌2型 溶菌酶释放蛋白 胞外蛋白因因子 融合表达 中和表位 |
| 收稿时间： | 2004-9-29 |
Cloning and Expression of a Fusion Protein of Muramidase-Released Protein and Extracellular Protein Factor of Streptococcus suis Type 2 and Its Protection |
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| LI Ming,HE Kong-wang,LU Cheng-ping.Cloning and Expression of a Fusion Protein of Muramidase-Released Protein and Extracellular Protein Factor of Streptococcus suis Type 2 and Its Protection[J].Scientia Agricultura Sinica,2005,38(6):1264-1269. | |
| Authors: | LI Ming HE Kong-wang LU Cheng-ping |
| Abstract: | 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. |
| Keywords: | Streptococcus suis type 2 Muramidase-released protein Extracellular protein factor Fusion expression Epitope |
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