停乳链球菌内蒙分离株mig基因的克隆和表达.pdfVIP

摘 要 停乳链球菌是引起奶牛乳房炎的主要病原菌，根据GenBank中登陆的停乳链 球菌表面蛋白MIG基因序列，设计一对引物，采用PCR的方法从临床分离的停乳 链球菌内蒙分离株基因组DNA中扩增出MIG基因，得到一条约1900 bp的片段。将 其连入PMD-19T载体中，经酶切，PCR及序列测定法进行鉴定。结果表明，MIG基 因与GenBank中停乳链球菌MIG基因 (AF354651)序列的同源性为95%。构建原核表 达载体PET-32 ａ(+)-MIG，随后转化到大肠杆菌BL21 (DH5a)中表达。表达产物进 行SDS分析后， 表达的重组蛋白大小为89 ku。将纯化的MIG重组蛋白免疫 家兔，用ELISA检测其血清抗体。用制备出的抗血清与链球菌全菌进行玻片凝集 试验，出现凝集颗粒。结果表明MIG重组蛋白具有较强的免疫原性，为停乳链球 菌疫苗的研制奠定基础。 关键词：乳房炎停乳链球菌；MIG 蛋白；克隆；原核表达；抗血清 Cloning and Prokaryotic Expression of MIG Gene Sequence of Streptococcus dysgalactiae from Strain Inner Mongolia Abstract Streptococcus dysgalactiae is the major pathogens of mastitis in dairy cows , S.dysgalactiae were derived from the mastitis Bovine in some areas of Inner Mongolia 。A pair of primers was designed according to Streptococcus dysgalactiae MIG surface protein gene sequences in GenBank .Subsequently,the MIG gene of 1900 bp was amplified by PCR.Then ,the gene was cloned into PMD- 19T vecto，and identified by digestion with restriction endonucleases ,PCR and sequencing. The DNA sequence analysis confirmed Streptococcus dysgalactiae in GenBank, MIG gene (AF354651) sequence homology of 95%. A porkaryotic expression pET-32a(+)-MIG was constructed .The recombinant plasmid was transformed into BL21(DH5 a)/pET system for expression .The recombinant protein was analyzed by SDS ， it had an approximate molecular mass of 89 ku. With the purified protein immune rabbits , and anti-MIG antibodies were measured by ELISA . With the antiserum prepared with whole bacteria Streptococcus slide agglutination test, agglutination particles appeared.The results show that the MIG fusion protein with strong immunogenicity for vaccine dysgalactiae basis.