柔嫩艾美耳球虫gam22基因重组质粒的免疫原性及保护效果

Immunogenicity and Protective Efficacy of Recombinant Plasmid Harboring Eimeria tenella gam22

为了评价重组质粒pSMART2b-Etgam22抗柔嫩艾美耳球虫的保护效果,本试验用PCR扩增获得Etgam22基因后连接至pSMART2b载体以构建重组质粒pSMART2b-Etgam22,用Western Blot分析重组质粒pSMART2b-Etgam22在293T细胞中的表达情况,用重组质粒pSMART2b-Etgam22免疫雏鸡,并检测抗体水平和细胞因子分泌情况,随后用柔嫩艾美耳球虫卵囊感染免疫的雏鸡,观察死亡情况,检测体重变化、卵囊数量、盲肠病变,通过抗球虫指数评价pSMART2b-Etgam22对柔嫩艾美耳球虫感染的保护效果。结果显示,本试验成功构建了重组质粒pSMART2b-Etgam22;Western Blot分析表明,该重组质粒可在细胞中表达融合蛋白,表达蛋白可被抗柔嫩艾美耳球虫阳性血清识别。与未免疫感染组相比,pSMART2b-Etgam22免疫雏鸡显著提升了抗体水平(P<0.01)及IL-2和IFN-γ分泌(P<0.01),显著增加了体重(P<0.01),显著减少了卵囊排出数量(P<0.05),显著减轻了盲肠病变(P<0.05)。根据存活率、相对增重率、盲肠病变计分、卵囊值计算得出的抗柔嫩艾美耳球虫指数为165。结果表明,pSMART2b-Etgam22具有良好的抗柔嫩艾美耳球虫保护效果。

This study investigated immunogenicity and protective efficacy of recombinant plasmid pSMART2b containing Eimeria tenella gam22 gene(Etgam22).Etgam22 gene was amplified by PCR and ligated into alphavirus replicon vector pSMART2b to yield recombinant plasmid pSMART2b-Etgam22.Etgam22 protein was expressed in 293T cells and analyzed by Western Blot.Chickens were immunized with recombinant plasmid pSMART2b-Etgam22,and the levels of anti-Etgam22 antibody,IL-2 and IFN-γ in serum were assessed.The protective efficacy of pSMART2b-Etgam22 against E.tenella challenge infection was evaluated by the anticoccidial index,chicken mortality,weight change,oocyst number and cecum lesion.The results showed that chickens immunized with recombinant plasmid pSMART2b-Etgam22 had significantly higher levels of anti-Etgam22 antibody(P<0.01),IL-2 and IFN-γin serum(P<0.01),higher body weight(P<0.01),lower oocyst production(P<0.05)and milder cecum lesions(P<0.05)than non-immunized controls.As measured by survival rate,relative weight gain rate,mean lesion scores of cecum and oocyst value,the anticoccidial index of the pSMART2b-Etgam22 group was calculated to be 165.The results indicating that pSMART2b-Etgam22 had a good protective efficacy.