鸡球虫多价多表位亚单位疫苗免疫原性分析及抗球虫感染的效果观察

Immunogenicity analysis and immune efficacy evaluation against Eimeria spp.of multivalent recombinant subunit vaccine

[目的]本试验旨在评估重组蛋白rN1T1L2E1和rN1T1L2E1I2作为亚单位疫苗候选抗原的潜力。[方法]应用PCR技术分别扩增NA4-1-TA4-1-LDH-2-EMCDPK-1与NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2基因,用LaserGene分析基因编码蛋白序列特性;表达重组蛋白rN1T1L2E1和rN1T1L2E1I2,经Western blot分析重组蛋白的免疫原性,采用流式细胞术检测蛋白免疫后鸡脾脏中CD^(+)_(4)和CD^(+)_(8) T淋巴细胞的比值,经间接ELISA法检测鸡血清中细胞因子水平和特异性IgG水平以分析重组蛋白的免疫原性。取柔嫩艾美耳球虫、毒害艾美耳球虫、巨型艾美耳球虫和堆型艾美耳球虫新鲜卵囊经口感染免疫后的雏鸡,统计平均增重、病变计分、盲肠内容物卵囊数等指标,计算各组抗球虫指数(ACI),评估重组蛋白对球虫感染的免疫保护效果。[结果]LaserGene分析显示重组蛋白拥有密集且连续的T细胞表位,抗原指数高;Western blot分析显示重组蛋白可被4种艾美耳球虫的多克隆抗体识别,重组蛋白rN1T1L2E1和rN1T1L2E1I2均可提高机体CD^(+)_(4)/CD^(+)_(3)和CD^(+)_(8)/CD^(+)_(3)细胞比例,提高血清中IFN-γ、IL-4、IL^(-1)7、TGF-β和IgG抗体水平。以上结果表明重组蛋白具有良好的反应原性和球虫抗原性。免疫重组蛋白后试验组鸡平均增重高于对照组,卵囊产量下降明显,肠道病变程度减轻,抗球虫指数提升。[结论]重组蛋白rN1T1L2E1和rN1T1L2E1I2可作为多价多表位亚单位抗球虫疫苗的候选抗原。

[Objectives]The experiment aimed to evaluate the potentialities of the recombinant proteins rN1T1L2E1 and rN1T1L2E1I2 as candidate antigens of recombinant subunit vaccine.[Methods]The NA4-1-TA4-1-LDH-2-EMCDPK-1 gene and NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 gene were cloned by PCR,the protein sequence characteristics encoded by the genes were analysed using LaserGene;the recombinant proteins rN1E1L2E1 and rN1E1L2E1I2 in prokaryotic cells were expressed.The immunogenicity of the recombinant proteins were analyzed by Western blot.The proportion of CD^(+)_(4) T lymphocytes and CD^(+)_(8) T lymphocytes in spleen of chickens was detected by flow cytometry.The levels of cytokines and specific IgG in chicken serum were detected by indirect ELISA.E.tenella,E.necatrix,E.maxima and E.acervulina oocysts were used to infect chickens by oral infection.The average weight gain,lesion score and the number of oocysts per gram of cecal contents in chickens were observed.The anticoccidiosis index(ACI)of each group was calculated to evaluate the protection effect of recombinant protein.[Results]LaserGene analysis showed that the two recombinant proteins had dense and continuous T-cell epitopes with high antigen index.Western blot analysis showed that the recombinant protein could be recognized by chicken serum against four kinds of avian coccidiosis above.The results showed that the subpopulations of the CD^(+)_(4) and CD^(+)_(8) T cells and the levels of serum IFN-γ,IL-4,IL^(-1)7,TGF-βand IgG increased significantly compared to the control by immunized with proteins rN1T1L2E1 and rN1T1L2E1I2.Vaccination with multivalent recombinant subunit vaccine significantly increased the weight gains,decreased the oocyst productions alleviated the enteric lesions and increased anticoccidial index,compared with the control group.[Conclusions]rN1T1L2E1 and rN1T1L2E1I2 can be used as candidate antigens for multivalent anticoccidiosis vaccines.