Stefin蛋白对人工感染豆状囊尾蚴家兔的免疫保护作用  

Immunoprotection of Stefin protein on rabbits artificially infected with Cysticercus pisiformis

作　　者：李甲 羊倩倩 王由森 肖琴 孙晓林[1] 王泽祥 LI Jia;YANG Qianqian;WANG Yousen;XIAO Qin;SUN Xiaolin;WANG Zexiang(College of Veterinary Medicine,Gansu A gricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《中国兽医科学》2024年第7期937-945,共9页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32160843)。

摘　　要：检测重组蛋白Cpstefin免疫家兔的抗体消长水平并验证其对豆状囊尾蚴感染的免疫保护效果。将本实验室保存的转化重组质粒pGEX-4T-Cpstefin的BL21(DE3)菌种活化,采用IPTG诱导表达后收集菌体进行SDS-PAGE分析鉴定后,运用GST琼脂糖树脂纯化Stefin蛋白,检测重组蛋白浓度。将测定好浓度的重组蛋白分别与弗氏佐剂、206佐剂、氢氧化铝佐剂按1∶1体积比充分乳化后免疫家兔。首次免疫300μg/只,加强免疫200μg/只,每次免疫间隔7 d,共免疫3次,并于第2次加强免疫1周后检测家兔外周血液抗体水平。免疫第30天每只家兔经口感染2000个豆状带绦虫虫卵,攻虫后第58天将家兔安乐死,计数囊荷数并计算各免疫组的减囊率。攻虫后每7 d耳缘静脉采血,检测血清抗体水平。结果显示,对各组家兔注射重组蛋白抗原后,免疫组家兔抗体水平开始升高,经口感染虫卵后,免疫组抗体水平有所下降,但D_(492 nm)值仍保持在2.50以上。剖检家兔计算减囊率,弗氏佐剂组、206佐剂组、氢氧化铝佐剂组减囊率分别为76.70%、66.02%和54.37%。结果表明,豆状囊尾蚴Cpstefin重组蛋白对实验感染豆状囊尾蚴家兔具有免疫保护作用。To monitor antibody dynamics in rabbits immunized with recombinant Cpstefin and verify its immunoprotective effect against Cysticercus pisiformis,the E.coli BL21(DE3)strain transformed with p GEX-4T-Cpstefin stored in the laboratory were activated,and the recombinant protein was induced by IPTG followed by SDS-PAGE analysis.The recombinant Stefin protein was purified by GST agarose resin.The purified recombinant protein emulsified with Freund’s adjuvant,206 adjuvant and aluminum hydroxide adjuvant in a 1︰1 volume ratio were respectively immunized into rabbits.The first immunization dose was 300μg per rabbit followed by booster immunization with 200μg per rabbit for three times.The interval of each immunization was set as 7 days,and the antibody level in peripheral blood of rabbits was detected one week after the second booster immunization.Immunization on the 30th day,each rabbit was orally challenged with 2000 eggs of Taenia pisiformis.On the 58th day after infection,rabbits were euthanatized and the number of C.pisiformis in each rabbit was counted and the reduction rate of C.pisiformis among rabbits in three immunized groups compared with the control group were calculated.At 58th day after infection,blood of rabbits were collected from auricular vein and the antibody dynamics of each rabbit was determined by ELISA.Post immunization,the antibody level of rabbits in three immunized groups continuously elevated.Although the antibody level of rabbits in three immunized groups declined post infection,the antibody level didn’t exhibit significant decrease and still maintained in a certain level.The reduction rate of C.pisiformis among rabbits in group Freund’s adjuvant,group206 adjuvant and group aluminum hydroxide adjuvant reached to 76.70%,66.02%and 54.37%,respectively.In conclusion,recombinant protein Cpstefin has immunoprotective effect against C.pisiformis in experimentally infected rabbits.

关 键 词：豆状囊尾蚴 半胱氨酸蛋白酶抑制剂Stefin 抗体消长 减囊率 免疫保护 

分 类 号：S852.734[农业科学—基础兽医学]