表达猪源链球菌类M-MRP融合蛋白基因工程菌的构建及免疫保护试验  

Construction of Recombinant Strain Expressing M-like-MRP Fusion Protein of Streptococcus from Pig and Its Immonoprotection Protection Test

作　　者：范红结[1] 陆承平[1] 

机构地区：[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

出　　处：《农业生物技术学报》2006年第1期22-26,共5页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划项目(No.1999011906)资助

摘　　要：利用PCR技术,分别扩增马链球菌兽疫亚种(Streptococcusequisubsp.zooepidemicus)ATCC35246株类Mszp基因583￣1063位碱基和猪链球菌(S.suis)2型HA9801株mrp基因298￣827位碱基的片段。通过在类M基因片段的3!端和mrp基因片段的5!端引物添加共同的SacⅠ酶切位点,利用T4连接酶连接,克隆至pET-32a(+)载体。将重组质粒转化大肠杆菌(Escherichiacoli)BL21,经IPTG诱导,可获得分子量约为60kD的融合蛋白。通过MRP和类M蛋白的多克隆抗体进行ELISA反应和免疫印迹分析表明:表达的融合蛋白同时具有类M蛋白和MRP的抗原性。用组氨酸亲和层析柱纯化的重组融合蛋白,和链球菌的全菌二联灭活疫苗分别免疫两组仔猪,重组蛋白的免疫保护率达60"。The 480 bp fragment of M-like（szp） gene and 529 bp fragment of mrp gene were amplified from the genomic DNA of Streptococcus equi subsp, zooepidemicus ATCC35246 strain and S.suis type 2 HA9801 respectively by PCR. These two gene fragments were fused by T4 ligarase and then the fusion gene was cloned into pET-32a （＋） in the proper orientation via restriction endonucleases BamHⅠ and H/ridⅢ. The recombinant plasmid was verified by restriction endonuclease analysis, nucleotide sequencing and double PCR, then transformed into its host Escherichia coli strain BL21 . The fusion gene could encod the recombinant fusion protein with 339 amino acid residues. SDS-PAGE and Western blotting analysis showed that the recombinant fusion protein had a molecular weight of 60 kD, and a positive reaction with the antiserum against MRP and M-like protein. ELISA analysis demonstrated that part of M-like protein and MRP existed in the fusion protein. Two groups of pigs were immunized, respectively with the purified recombinant protein and the inactivated combined vaccine, and the purified recombinant protein offered a 60% protection against the challenge of S.equ/subsp. zooepidemicus ATCC35246 and S.su/s type 2 HA9801.

关 键 词：类M基因 MRP基因 基因融合 表达 

分 类 号：S188[农业科学—农业基础科学]