猪链球菌2型溶血素成熟蛋白的原核表达及其生物学特性与免疫保护性  

Prokaryotic expression,bioactivity and immuonoprotection test of suilysin mature protein of Streptococcus Suis type 2

作　　者：倪艳秀[1] 何孔旺[1] 周俊明[1] 汪伟[1] 吕立新[1] 俞正玉[1] 茅爱华[1] 温立斌[1] 张雪寒[1] 李彬[1] 胡屹屹[1] 郭容利[1] 王小敏[1] 陆承平[2] 

机构地区：[1]江苏省农业科学院兽医研究所,农业部兽用生物制品工程技术重点实验室,国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《江苏农业学报》2013年第2期335-340,共6页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31072155);江苏省自然科学基金重点项目(BK2010068);江苏省农业科技自主创新探索性研究项目[CX(11)4040]

摘　　要：根据猪链球菌2型溶血素(SLY)的基因序列设计引物,以江苏分离株SS2-1的基因组为模板,扩增除信号肽序列外的溶血素成熟蛋白基因,并克隆到表达载体pET-32α(+)中,转化大肠杆菌BL21(DE3)。重组菌经1 mmol/L IPTG诱导,在37℃下4 h时表达产物(分子量约为7.2×104)达到高峰,表达量约占菌体总蛋白质的20%,表达产物在上清和包涵体中各占一半。将上清经镍柱亲和层析纯化,纯化蛋白质经Western blotting鉴定呈阳性,对绵羊红细胞、鸡红细胞的溶血效价分别为1.707×105HU/mg、4.267×104HU/mg,并可引起HEp-2和Vero细胞的颗粒增多、圆缩和脱落。纯化蛋白质对BALB/c小鼠的免疫保护率为83.3%(5/6)。这为进一步研究SLY致病机理和研制猪链球菌新型疫苗奠定了基础。A pair of specific primers was designed according to suilysin gene of Streptococcus suis type 2. Using the genomic DNA of SS2-1 isolated from Jiangsu province as the template, the suilysin mature protein gene in addition to the signal pePtide sequence was amplified and was cloned into expression vector pET-32α（ ＋ ）. The recombinant plasmid was transformed into the Escherichia. coli strain BL21 （DE3）. After induced by 1 mmo]/L IPTG at 37 ℃ for 4 h, the fused protein with molecular weight about 7.2×10^4 was at the highest level. The yield of the expression product was about 20% of total bacterial proteins. Western blotting revealed that the purified protein contained antigenic derteminant of SLY. The hemolytic titers of the protein to sheep red cell and chick red cell attained 1. 707×10^5 HU/mg and 4. 267× 10^4 HU/mg, respectively. The purified protein could cause Vero or HEp-2 cell grain increase, round shrink and fall. The protection rate of purified recombinant protein rSLY to BALB/ c mice was 83.3% （5/6）. This study established a solid foundation for further study on pathogenesis of suilysin （SLY） and new type vaccine development of Streptococcus suis.

关 键 词：猪链球菌2型 溶血素成熟蛋白 原核表达 生物学特性 免疫保护性 

分 类 号：S859.797[农业科学—临床兽医学]