摘要
目的制备堆型艾美耳球虫(Eimeria acervulina)重组亚单位疫苗,并评价其免疫效果。方法将堆型艾美耳球虫的保护性抗原基因cSZ1克隆至酵母表达载体pPICZαA,构建重组表达质粒pPICZαA-cSZ1,转化毕赤酵母菌株GS115,构建重组菌GS115-pPICZαA-cSZ1,经甲醇诱导表达重组蛋白cSZ1。按20、40、60μg/只(低、中、高剂量)经雏鸡肌肉注射重组蛋白,7 d后进行加强免疫,加强免疫7 d后经口感染E.acervulina孢子化卵囊,剂量为1×10^(4)个/只,同时设阳性对照组(未免疫仅攻虫)和阴性对照组(未免疫未攻虫)。攻虫后7 d检测雏鸡的抗球虫指数(anticoccidial index,ACI);初次免疫后7 d及加强免疫后7 d,经雏鸡心脏采血,分离血清,间接ELISA法检测血清抗体水平;攻虫后7 d,无菌取雏鸡脾脏,CCK-8法检测T淋巴细胞增殖情况。结果经双酶切及测序鉴定,证明重组表达质粒构建正确;经菌落PCR及测序鉴定,证明重组菌构建正确。重组蛋白cSZ1的相对分子质量约19000,可与鼠抗His标签单克隆抗体发生特异性结合。低、中、高剂量免疫组的ACI分别为171.25、176.42、174.33;与阴性对照组比较,低、中、高剂量免疫组雏鸡于初次免疫后7 d及加强免疫后7 d,血清抗体水平明显升高(P<0.01),攻虫后7 d,T淋巴细胞增殖率显著升高(P<0.01)。结论制备的堆型艾美耳球虫重组亚单位疫苗对雏鸡有较好的免疫效果。
Objective To prepare recombinant Eimeria acervulina subunit vaccine and evaluate its immune effect.Methods The protective antigen gene cSZ1 of E.acervulina was cloned into yeast expression vector pPICZαA,and the constructed recombinant plasmid pPICZαA-cSZ1 was transformed to Pichia pastoris GS115 strain.The obtained recombinant P.pastoris GS115-pPICZαA-cSZ1 was induced with methanol.Chicks were immunized i.m.with the expressed recombinant protein cSZ1 at low(20μg),moderate(40μg)and high(60μg)dosages respectively,and boosted 7 d later.Seven days after the booster immunization,the chicks were challenged with 1×10^(4) sporulated oocysts of E.acervulina by oral route,using those challenged without immunization as positive control and those without challenge or immunization as negative control.The anticoccidial index(ACI)was determined 7 d after challenge.Serum samples were collected 7 d after primary immunization and 7 d after booster immunization,and determined for antibody level by indirect ELISA.The spleens of chicks were collected aseptically 7 d after challenge and determined for T lymphocyte proliferation rate by CCK-8 method.Results Restriction analysis and sequencing proved that recombinant plasmid pPICZαA-cSZ1 was constructed correctly.Colony PCR and sequencing proved that recombinant P.pastoris GS115-pPICZαA-cSZ1 was constructed correctly.The expressed recombinant protein cSZ1,with a relative molecular mass of about 19000,showed specific binding to mouse monoclonal antibody against His tag.The ACIs of chicks immunized with cSZ1 at low,moderate and high dosages were 171.25,176.42 and 174.33 respectively.As compared with those in negative control group,the serum antibody levels of chicks 7 d after primary immunization and 7 d after booster immunization with cSZ1 at low,moderate and high dosages increased significantly(P<0.01),while the T lymphocyte proliferation rate 7 d after challenge increased significantly(P<0.01).Conclusion Recombinant E.acervulina subunit vaccine was prepared,which showed good
作者
毕天奇
肇英池
马丹
程淑琴
王晓岑
李新
李建华
张西臣
宫鹏涛
张楠
张媛媛
BI Tian-qi;ZHAO Ying-chi;MA Dan;CHENG Shu-qin;WANG Xiao-cen;LI Xin;LI Jian-hua;ZHANG Xi-chen;GONG Peng-tao;ZHANG Nan;ZHANG Yuan-yuan(College of Veterinary Medicine,Jilin University,Changchun 130062,Jilin Province,China;不详)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2022年第7期790-794,801,共6页
Chinese Journal of Biologicals
基金
吉林省科技发展计划项目(20190301089NY,20190103075JH)
石家庄市科学技术研究与发展计划项目(191500283A)。
