摘要
根据已发表的马链球菌兽疫亚种MGCS10565酮基转移酶(transketolase)的基因序列,设计并合成引物。以ATCC35246株基因组DNA为模板,通过PCR技术,扩增出目的基因并定向克隆至表达载体pET-28a(+)中,然后将重组质粒转化入大肠杆菌BL21(DE3)中,分析并纯化表达产物。选用ICR小鼠作为实验动物模型,以纯化的重组融合蛋白通过皮下注射途径免疫小鼠,并用间接ELISA法监测小鼠血清中的抗体效价。结果表明重组蛋白免疫小鼠后能产生有效的免疫应答,血清中抗体水平有明显的升高。加强免疫2周后,以5LD50的ATCC35246强毒株攻击免疫组及对照组,结果免疫组小鼠的保护率可达37.5%。表明原核表达产物免疫ICR小鼠,可使其对同源菌株攻击产生一定的保护作用,在亚单位疫苗研制中具有潜在的应用价值。
Specific primers were designed according to the transketolase gene of Streptococcus equi ssp. zooepidemicus （ SEZ ） strains MGCS10565. The target gene was amplified from genomic DNA of SEZ strain ATCC35246 by PCR, and inserted into the expression vector pET-28a （＋） to construct the recombinant plasmid pET-28a-TK. The expression product of pET-28a-TK was analyzed by western blot. ICR mice were immunized with purified recombinant protein through hypodermic inoculation. The results of indirect ELISA showed that effective immunological responses were induced in mice, and serum antibody levels were significantly increased. Two weeks after the booster immunization, all groups were challenged intraperitoneally with 5LD50 of the virulent strain ATCC35246. The results showed that the protective rate of immunized mice was 37.5%. So the recombinant protein showed the immune protective potency in mice against SEZ infections and it is a promising candidate for the subunit vaccine development in the future.
出处
《畜牧与兽医》
北大核心
2012年第8期8-12,共5页
Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31172319)
关键词
马链球菌兽疫亚种
酮基转移酶
原核表达
免疫保护
Streptococcus equi ssp. zooepidemicus
transketolase
prokaryotic-expression
immune protection
