马链球菌兽疫亚种类M蛋白的基因克隆、序列分析及其在猪源链球菌的检测  

Cloning and Sequence Analysis of M-like Gene of Streptococcus equi subsp. zooepidemicus and The Detection of M-like Gene of Streptococci from Pigs

作　　者：范红结[1] 陆承平[1] 唐家琪[1] 

机构地区：[1]南京农业大学畜禽疫病诊断与免疫重点开放实验室,南京210095

出　　处：《微生物学报》2004年第5期617-620,共4页Acta Microbiologica Sinica

基　　金：国家"973项目"(G19990 1190 6)~~

摘　　要：根据已发表的马链球菌兽疫亚种 (Streptococcusequisubsp .zooepidemicus)马源株的类M蛋白基因序列 ,设计和合成一对引物 ,以兽疫亚种猪源ATCC35 2 4 6株的基因组DNA为模板 ,通过PCR技术 ,扩增出类M蛋白基因并定向克隆至表达载体pET_32a(+)中 ,测定其序列 ,GenBank接收号为AY2 6 3781。类M蛋白基因含一个完整的开放阅读框 ,全长为 1137bp ,编码 379个氨基酸残基。经DNAStar软件分析 ,ATCC35 2 4 6株类M蛋白基因与兽疫亚种马源W6 0株、马亚种的类M蛋白基因及A群化脓链球菌的M蛋白基因的同源性分别为 86 9%、30 8%、2 9 4 % ;推导的氨基酸序列的同源性分别为 84 3%、2 1 9%、2 3 4 %。但ATCC35 2 4 6株类M蛋白的C末端细胞膜锚定区与M蛋白、马亚种类M蛋白高度同源。用上述设计的引物进行PCR试验 ,检测 34株猪源链球菌类M蛋白基因 ,发现所有 16株C群猪源链球菌均能检测出类M蛋白基因 ,而所有猪链球菌 (Streptococcussuis) 1型和 2型菌株及S群、B群、D群链球菌共 13株均不能检出类M蛋白基因 ,而 5株未鉴定的猪源链球菌中 3株能检测出类M基因。Streptococcus equi subsp. zooepidemicus,a lancefield group C \%Streptococcus,\% causes a variety of serious infections in several animal hosts, including meningitis, pneumonia, septic arthritis and mastitis.The antiphagocytic M like protein in cell surface of S. equi subsp. zooepidemicus is a major virulence factor and protective antigen. In this paper, the M-like gene was amplified from genomic DNA of S. equi subsp. zooepidemicus ATCC35246 strain by polymerase chain reaction (PCR). Then the amplified fragment was cloned in the proper orientation into the site between BamHⅠand SacⅠof pET32-a(+) via restriction endonuclease BamHⅠand SacⅠ.The recombinant plasmid was verified by restriction endonuclease analysis and nucleotide sequencing. Nucleotide sequencing analysis revealed one open reading frame of 1137 in M-like gene, translation of this open reading frame revealed a protein of 379 amino acids. The M-like gene showed 86.9% nucleotide acids homology with M-like gene of S. equi subsp. zooepidemicus W60 strain, 28% with M-like gene of S. equi subsp. equi and 29.4% with M protein gene of group A \%Streptococcus.\% The M-like protein of ATCC35246 strain showed 84.3%, 21.9%, 23.4% amino acid homology with S. equi subsp. zooepidemicus W60 strain, S. equi subsp. equi and group A \%Streptococcus. 34 Streptococcus\% isolated strains from pigs were detected for M-like gene by PCR, the results showed all 16 strains of group C \%Streptococcus\% strain from pigs contained M-like gene, the M-like gene weren't detected in other 13 strains of R, S,B,D group,but 3 strains contained the M-like gene in the other 5 no identified isolated strains of \%Streptococcus.\%

关 键 词：马链球菌兽疫亚种 类M蛋白基因 克隆 检测 

分 类 号：S852.61[农业科学—基础兽医学]