堆型艾美耳球虫cSZ1基因在减毒沙门氏菌的表达及免疫保护效果研究  

IMMUNOGENIC CHARACTERIZATIONS OF EIMERIA ACERVULINA cSZ1 GENG EXPRESSED IN ATTENUATED SALMONELLA TYPHIMURIUM

作　　者：覃宗华[1] 谢明权[1] 蔡建平[1] 艾哈迈德[1] 叶秀华[1] 

机构地区：[1]广东省农业科学院兽医研究所,广州510640

出　　处：《寄生虫与医学昆虫学报》2005年第1期6-13,共8页Acta Parasitologica et Medica Entomologica Sinica

基　　金：广东省自然科学基金 (No .0 0 0 14 5 );国家"863"计划 (No .2 0 0 2AA2 413 3 1)资助

摘　　要：用PCR方法扩增堆型艾美耳球虫广东株子孢子表面抗原基因cSZ1的阅读框架 (ORF) ,与质粒表达载体pYA3342连接后转化大肠杆菌E-coliX6 2 12 ,获得阳性重组质粒后将其电转化至减毒鼠伤寒沙门氏菌(Salmonellatyphimurium)X4 5 5 0。经IPTG诱导获得了cSZ1基因在减毒S typhimurium的高效表达 ,表达产物占菌体总蛋白的 9 2 % ,重组蛋白分子量约为 19kDa。将重组减毒S typhimurium分别以 10 8或 10 9CFU 鸡经口免疫 4日龄岭南黄肉鸡 ,免疫后两周血清中可检测到抗cSZ1的特异性IgG ,3周时抗体水平达到峰值 ;2 5日龄时可在肠道检测到特异性IgA的分泌。免疫后 3周 ,试验鸡分别经口攻击感染 5 0 0个E acervulina孢子化卵囊 ,结果显示免疫组与非免疫组有相似的排卵囊曲线 ;计数攻虫感染后第 3 5～ 9 5天卵囊总产量 ,2个免疫组分别能降低 2 5 . 1%和 4 6 . 4 %的卵囊产生。The ORF of the surface antigen gene,cSZ1 was amplified from Eimeria acervulina(GD strain) by PCR and ligated to plasmid pYA3342 to construct a recombinant plasmid pYA3342-cSZ1.Then this recombinant plasmid was transformed to Escherichia coli X6212.The positive recombinant plasmid identified by endonuclease digestion and PCR methods was electroporated into attenuated S.typhimurium X4550.The recombinant cSZ1 was expressed in Salmonella induced with 1mmol/L IPTG.The recombinant cSZ1 protein with molecular weight about 19kDa,about 9.2% of total bacterial proteins,could not be probed by hyperimmune serum against E.acervulina(GD) by Western Blotting.However,specific serum IgG against recombinant sCZ1 was detected in chickens vaccinated with 108 or 109CFU recombinant Salmonella 2 weeks post immunization (PI) and its peak appeared 3 weeks PI.Specific IgA in intestine was also observed 3 weeks PI.These immunized chicknes were challenged with 500 homologous sporulated occysts 3 weeks PI,and could reduce the oocyst output by 25.1% or 46.4% respectively in low or high vaccinating dosage compared with the control.

关 键 词：堆型艾美耳球虫 减毒沙门氏菌 效果研究 免疫保护 大肠杆菌E.coli 减毒鼠伤寒沙门氏菌 表面抗原基因 质粒表达载体 特异性IgG PCR方法 孢子化卵囊 重组质粒 高效表达 IPTG 表达产物 重组蛋白 抗体水平 子孢子 广东株 电转化 

分 类 号：Q785[生物学—分子生物学]