摘要
为了确定重组质粒pPIC9-IL-2-SO7在毕赤酵母系统中的表达及重组蛋白SO7的免疫保护力,采用RT-PCR方法克隆E.tenella河北株SO7基因并测序,将目的基因与IL-2及真核表达载体pPIC9串联,构建真核表达载体pPIC9-IL-2-SO7,转染毕赤酵母细胞GS115,SDS-PAGE电泳及Western blot方法验证其在毕赤酵母系统中的表达。将纯化后的重组蛋白SO7分别在雏鸡7,21日龄以50,100,150μg/只进行免疫,28日龄时,除阴性对照组外的其他试验组口服接种E.tenella孢子化卵囊8×10^4个/只,测定各组存活率、相对增重率、盲肠病变值和卵囊值,并计算抗球虫指数(ACI)。结果显示,阳性重组子具有2条带,分别为2 200,1 100 bp,表明成功将重组质粒pPIC9-IL-2-SO7转染毕赤酵母细胞GS115,Western blot检测证明表达的蛋白具有免疫原性;3个免疫组的ACI分别比阳性对照组提高了29.66%,33.33%和19.72%,其中以免疫剂量为100μg/只时,抗球虫效果最好。
In order to determine the expression of recombinant plasmid pPIC9-IL-2-SO7 in Pichia pastoris system and the immunoprotective effect of recombinant protein SO7,the SO7 gene of E.tenella Hebei strain was cloned by RT-PCR and sequenced.IL-2 and eukaryotic expression vector pPIC9 were ligated in tandem to construct eukaryotic expression vector pPIC9-IL-2-SO7,transfected into Pichia pastoris cell GS115,SDS-PAGE electrophoresis and Western blot method to verify its expression in Pichia pastoris system.The purified recombinant protein SO7 was immunized with chicks at 50,100 and 150 μg/only at 7 and 21 days of age,and at the age of 28 days,other test groups except the negative control group were orally inoculated with E.tenella sporulated oocysts 8×10^4/each chicken,the survival rate,relative weight gain rate of cecal lesions and oocysts were measured,and the anticoccidial index(ACI) was calculated.The results showed that the positive recombinants had 2 bands,2 200 bp and 1 100 bp,respectively,indicating that the recombinant plasmid pPIC9-IL-2-SO7 was successfully transfected into Pichia pastoris cell GS115,and the expressed protein was confirmed by Western blot assay.The ACI of the three immunized groups increased by 29.66%,33.33% and 19.72%,respectively,and the anticoccidial effect was the best when the immunization dose was 100 μg/only.
作者
闫艳娟
李蕴玉
张东林
李佩国
庞洪泽
张香斋
YAN Yan-juan;LI Yun-yu;ZHANG Dong-lin;LI Pei-guo;PANG Hong-ze;ZHANG Xiang-zhai(Hebei Province Key Laboratory of Veterinary Preventive Medicine,Hebei Normal University of Science&Technology,Qinhuangdao,Hebei 066600,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2020年第10期1960-1964,共5页
Chinese Journal of Veterinary Science
基金
河北省现代农业产业技术体系蛋肉鸡产业创新团队岗位资助项目(HBCT2018150206)
秦皇岛市科技支撑计划资助项目(201803B014)。
