马链球菌兽疫亚种类M基因和猪链球菌2型mrp基因片段的融合表达及仔猪免疫试验  

Fusion expression of Streptococcus equi subsp. zooepidemicus M-like gene fragment and S.suis type 2 mrp gene fragment and evaluation of its immune protection in piglets

作　　者：范红结[1] 陆承平[1] 唐家琪[2] 

机构地区：[1]南京农业大学农业部畜禽疫病诊断与免疫重点开放实验室,江苏南京210095 [2]南京军区军事医学研究所,江苏南京210002

出　　处：《南京农业大学学报》2003年第4期78-81,共4页Journal of Nanjing Agricultural University

基　　金：国家 973项目子课题 (G19990 1190 6)

摘　　要：将含猪链球菌 2型mrp及马链球菌兽疫亚种类M基因融合片段的重组质粒转化大肠杆菌BL 2 1,经异丙基硫代 β D 半乳糖苷 (IPTG)诱导 ,表达了相对分子质量为 6 0 0 0 0左右的融合蛋白。利用组氨酸亲和层析柱 ,获得了纯化的融合蛋白 ,质量浓度为 4 2 μg·mL-1。以纯化的融合蛋白、宿主菌BL 2 1经IPTG诱导表达后的超声波破碎上清、马链球菌兽疫亚种ATCC 35 2 4 6株和猪链球菌 2型HA 980 1株制备的二联灭活苗为免疫原 ,分别免疫 30头仔猪。初次免疫 1月后 ,以同样的剂量再次免疫 ,3周后以 5×LD50 的ATCC 35 2 4 6和HA 980 1强毒株进行攻击。结果 ,纯化融合蛋白免疫组的免疫保护率达 6 0 % ,BL 2 1上清免疫组的免疫保护率为 5 0 % ,全菌二联灭活菌苗的免疫保护率达 90 %。Streptococcus suis type 2 and S.equi subsp. zooepidemicus are the major pathogens in swine in China.A recombinant plasmid harboring 484 bp fragment of Streptococcus equi subsp zooepidemicus M-like protein gene and 528 bp fragment of S.suis type 2 mrp gene was transformed into its host strain BL21.The fusion gene encoded the recombinant protein of 339 amino acid.The expression product was purified by his-nitrilotriacetic acid affinity chromatography and had a molecular weight of 60 000 by SDS-PAGE analysis.Thirty non-immunized piglets were divided into three groups.Two groups of piglets were subcutaneously immunized,respectively,with the purified recombinant protein and the supernatant protein of the recombinant plasmid-containing E.coli BL21 culture subjected to IPTG induction and ultrasonic treatment,both at a protein concentration of 4.2 μg·mL -1 with white oil as adjuvant.Piglets of the third group were immunized with the inactivated vaccine with Streptococcus equi subsp.zooepidemicus M-like gene fragment and Streptococcus suis type 2 as control.All the immunized animals were challenged with the corresponding virulent bacterial strains after the second vaccination.The inactivated vaccine had a protection rate of 90%,while the purified protein offered a 60% protection against the challenge.The protective rate was 50% for the group of animals receiving ultrasonic treated supernatant protein.All piglets without vaccination died within one week.

关 键 词：马链球菌兽疫亚种M基因 猪链球菌2型mrp基因 融合表达 仔猪 免疫试验 链球菌病 二联疫苗 

分 类 号：S858.28[农业科学—临床兽医学]