摘要
【目的】通过对柔嫩艾美耳球虫(E.tenella)Profilin基因进行原核表达和纯化,探明重组Profilin蛋白对鸡的免疫保护效果。【方法】设计合成Profilin基因特异性引物,提取E.tenella孢子化卵囊总RNA,反转录成cDNA,用PCR扩增获得Profilin基因后连接至pET-32a(+)载体构建重组质粒pET-32a(+)-Profilin,在IPTG诱导下进行表达并纯化。用重组Profilin蛋白免疫雏鸡,随后经口服感染柔嫩艾美耳球虫卵囊,测定和统计雏鸡存活率、平均增重、血便计数、每克粪便卵囊数、卵囊产量、盲肠病变,计算抗球虫指数,观察盲肠组织病理学变化并测定血清中IL-2、IL-10、TNF-α、sIgA的水平以评估重组Profilin蛋白的免疫保护效果。【结果】成功表达了重组Profilin蛋白,大小为39.1 kDa。免疫保护试验表明:Profilin重组蛋白组鸡只存活率、鸡只血便数和OPG相对于感染不免疫攻虫组显著减少(P<0.05);50μg重组蛋白免疫组的体增重效果最好,达93%,卵囊产量最少,ACI效果最好;100μg重组蛋白免疫组的盲肠损伤最小,而免疫器官指数未见显著差异(P>0.05)。在攻虫后7 d,感染不免疫攻虫组血清中IL-2、TNF-α、IL-10以及sIgA水平相比于空白对照组均极显著升高(P<0.01),而重组蛋白免疫组血清中IL-2、TNF-α、IL-10以及sIgA水平极显著低于攻虫不免疫组(P<0.01)。【结论】柔嫩艾美耳球虫重组Profilin蛋白可减少感染雏鸡的增重损失和卵囊排出,引发雏鸡体内的细胞免疫和体液免疫,具有一定的免疫保护效果。
[Objective]In this study,we investigated the immunoprotective effect of recombinant Profilin protein on chickens by prokaryotic expression and purification of E.tenella Profilin gene.[Methods]The Profilin gene specific primers were designed and synthesized,the total RNA of E.enella spore oocyst was extracted and reverse-transcribed into cDNA.Profilin gene was obtained by PCR amplification and then connected to pET-32a(+)vector to construct the recombinant plasmid pET-32a(+)-Profilin.It was expressed and purified under IPTG induction.Survival rate,mean weight gain,blood stool count,number of oocysts per gram of faeces(OPG),oocysts production,cecal lesions,and anti-coccidia index(ACI)were measured and calculated for chickens immunized with recombinant Profilin protein,followed by oral administration of Eimeria tender oocysts.The histopathological changes of cecum were observed and the levels of IL-2,IL-10,TNF-αand sIgA in serum were determined to evaluate the immunoprotective effect of recombinant Profilin protein.[Results]Recombinant Profilin protein with a size of 510bp was successfully expressed.Immune protection test showed that the survival rate,blood stool count and OPG of chickens in Profilin recombinant protein group were significantly decreased compared with those in non-immune attack group(P<0.05).The 50μg recombinant protein immunogroup had the best body weight gain at 93%,the least oocyst production,and the best ACI results.The 100μg recombinant protein-immunized group showed the least damage to the cecum,whereas no significant difference was observed in the immune organ index(P>0.05).At7days after attack,serum levels of IL-2,TNF-α,IL-10 and sIgA in the non-immune attack group were significantly higher than those in the blank control group(P<0.01).The levels of IL-2,TNF-α,IL-10 and sIgA in the recombinant protein immune group were significantly lower than those in the non-immune group(P<0.01).[Conclusion]Recombinant Profilin protein can induce cellular immunity and humoral immunity in the body,and shows good anti-coccidial effect in E.tenella infection.
作者
刘倩琳
李翌琳
周爽
武欣欣
刘永宁
段思彰
安健
张建军
LIU Qianlin;LI Yilin;ZHOU Shuang;WU Xinxin;LIU Yongning;DUAN Sizhang;AN Jian;ZHANG Jianjun(College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China)
出处
《北京农学院学报》
2024年第2期66-72,78,共8页
Journal of Beijing University of Agriculture
基金
2022年北京农学院学位与研究生教育改革与发展项目(2022YJS029)。
作者简介
第一作者:刘倩琳,女,硕士研究生,主要从事畜禽寄生虫病防治研究,E-mail:455390797@qq.com;并列第一作者:李翌琳;通信作者:张建军,博士,副教授,主要从事动物疾病病理、畜禽寄生虫病免疫预防研究,Tel:010-80795593,E-mail:zhjj339@163.com。
