鸡柔嫩艾美耳球虫5种免疫调节型DNA疫苗免疫保护效果的比较
摘要
鸡球虫病给养禽业所带来的经济损失极为严重。目前，应用药物防治遇到了球虫耐药性和药物残留的问题，大大限制了养禽业发展，球虫防治需要寻求新的途径。实践证明，激发宿主细胞免疫反应来防治鸡球虫病是更有效的途径之一。作为第三代疫苗的DNA疫苗的一个显著优势是刺激机体产生广泛的细胞免疫反应和持久的免疫记忆，尤其应用特定的球虫保护性抗原联合一些细胞因子，构建调节型DNA疫苗，在球虫病防治中具有诱人的应用前景。为此我们选择球虫的抗原MZ5-7基因与鸡细胞因子IFN-γ、IL-18基因，通过DNA重组技术将球虫的抗原基因分别和鸡细胞因子基因串联为一体，插入到真核表达载体pcDNA4.0中，分别构建了pcDNA4.0-MZ5-7-IFN-γ、pcDNA4.0-MZ5-7-IL-18两种免疫调节型核酸疫苗，同时与pcDNA4.0-MZ5-7、pcDNA4.0-MZ5-7-IL-2、pcDNA4.0-MZ5-7-IL-17三种免疫调节型核酸疫苗进行动物免疫保护性试验比较这些DNA疫苗的免疫保护效果。
利用本实验室构建好的柔嫩艾美耳球虫(E．tenella)MZ5-7原核表达载体pET28b-MZ5-7进行诱导表达，并对表达蛋白进行纯化后注射大鼠，制备大鼠E．tenella MZ5-7蛋白抗血清。将制备的MZ5-7蛋白血清用于MZ5-7基因疫苗在体内蛋白表达的检测。用特异性引物对MZ5-7基因疫苗在体内表达进行RT-PCR检测。结果证明构建的基因疫苗在体内能很好地转录表达，说明构建的疫苗可用于动物保护性实验研究。
动物保护性实验分为非感染非免疫对照组，感染非免疫对照组，pcDNA4.0空质粒组，pcDNA4.0-MZ5-7，pcDNA4.0-MZ-IL-18，pcDNA4.0-MZ-IL-17，pcDNA4.0-MZ-IL-2，pcDNA4.0-MZ-IFN-γ组。每组30只实验动物鸡。制备好的基因疫苗经肌肉于14日龄、21日龄两次注射雏鸡，28日龄经口接种新鲜的柔嫩艾美耳球虫孢子化卵囊。35日龄扑杀，分别作盲肠病变记分、克盲肠粪便卵囊数(OPG)、增重、存活率几个指标测，以抗球虫指数(ACI)综合判定其保护力的大小。结果显示，pcDNA4.0-MZ-IL-18，pcDNA4.0-MZ-IL-2，pcDNA4.0-MZ-IFN-γ具有明显的保护效果，其ACI分别为188.45、184.47、184.99。
Avain coccocidiosis causes severe economic loss in the poultry industry. Theemergence of drug-resistant parasites and the drug residues problem restricted thedevelopment of poultry industry so that a new approach to prevent avain coccocidiosisshould be found. It has been approved that stimulating the cell immuno-reaction of the hostis an effective approach to prevent avain coccocidiosis. A prominent advantage of DNAvaccine as a third generation of vaccine is that it can induce abroad cell immuno-reactionand form perdured immune memory. Particularly, the immune regulative DNA vaccinewhich was constructed by special antigen together with some cytokine genes has moreeffect and prospect in the anti- coccocidiosis practice. In this investigation we inserted theMZ5-7 antigenic gene of Eimeria tenella ligated with the chicken cytokine IFN-γ、IL-18into the vector pcDNA4.0 to construct immuno-regulative DNA vaccinepcDNA4.0-MZ5-7-IFN-γ、pcDNA4.0-MZ5-7-IL-18. The protective effects the abovementioned DNA vaccine together with pcDNA4.0-MZ5-7、pcDNA4.0-MZ5-7-IL-2、pcDNA4.0- MZ5-7-IL-17 were tested in the protective experiment of chickens against thechallenge of Eimeria tenella
IPTG was used to induce the expression of pET28b-MZ5-7 constructed in ourlabouratory. Purified MZ5-7 protein was used to immune rats for E. tenelIa MZ5-7anti-serum. The anti-serum was used to check the expression of MZ5-7 DNA vaccine inchicken by western-blot. Designed special primers were used to check the transcription ofMZ5-7 DNA vaccine in chicken by RT-PCR. The results showed that the DNA vaccinescould be well transcripted and expressed in chicken and they could be used to animalprotective experiment.
The chicken of protective experiment were divided into eight groups: pcDNA4.0、pcDNA4.0-MZ5-7、pcDNA4.0-MZ5-7-IL-18、pcDNA4.0-MZ5-7-IL-17、pcDNA4.0-MZ5-7-IL-2、pcDNA4.0-MZ5-7-IFN-γ、Unimmunized-unchallenged controlgroup、unimmunized-challenged control group. Each group had 30 chicken and were injected with vaccine through chest muscle. The vaccine was inoculated at 14 days and 21days of age and each chicken was challenged with 5×10~4 Eimeria tenelIa sporulated oocytesat 28 days of age of chicken. All chicken were killed at 35 days of age and the protectiveeffects were estimated by the index of lesion score of caecum, OPG, average body weightgain, anticoccidia index (ACI), The results showed that the immuno-regulative DNAvaccine pcDNA4.0-MZ5-7-IL-18、pcDNA4.0-MZ5-7-IL-2 pcDNA4.0-MZ5-7-IFN-γhadgood protective effects. Separately their ACIs are 188.45、184.47、184.99.
利用本实验室构建好的柔嫩艾美耳球虫(E．tenella)MZ5-7原核表达载体pET28b-MZ5-7进行诱导表达，并对表达蛋白进行纯化后注射大鼠，制备大鼠E．tenella MZ5-7蛋白抗血清。将制备的MZ5-7蛋白血清用于MZ5-7基因疫苗在体内蛋白表达的检测。用特异性引物对MZ5-7基因疫苗在体内表达进行RT-PCR检测。结果证明构建的基因疫苗在体内能很好地转录表达，说明构建的疫苗可用于动物保护性实验研究。
动物保护性实验分为非感染非免疫对照组，感染非免疫对照组，pcDNA4.0空质粒组，pcDNA4.0-MZ5-7，pcDNA4.0-MZ-IL-18，pcDNA4.0-MZ-IL-17，pcDNA4.0-MZ-IL-2，pcDNA4.0-MZ-IFN-γ组。每组30只实验动物鸡。制备好的基因疫苗经肌肉于14日龄、21日龄两次注射雏鸡，28日龄经口接种新鲜的柔嫩艾美耳球虫孢子化卵囊。35日龄扑杀，分别作盲肠病变记分、克盲肠粪便卵囊数(OPG)、增重、存活率几个指标测，以抗球虫指数(ACI)综合判定其保护力的大小。结果显示，pcDNA4.0-MZ-IL-18，pcDNA4.0-MZ-IL-2，pcDNA4.0-MZ-IFN-γ具有明显的保护效果，其ACI分别为188.45、184.47、184.99。
Avain coccocidiosis causes severe economic loss in the poultry industry. Theemergence of drug-resistant parasites and the drug residues problem restricted thedevelopment of poultry industry so that a new approach to prevent avain coccocidiosisshould be found. It has been approved that stimulating the cell immuno-reaction of the hostis an effective approach to prevent avain coccocidiosis. A prominent advantage of DNAvaccine as a third generation of vaccine is that it can induce abroad cell immuno-reactionand form perdured immune memory. Particularly, the immune regulative DNA vaccinewhich was constructed by special antigen together with some cytokine genes has moreeffect and prospect in the anti- coccocidiosis practice. In this investigation we inserted theMZ5-7 antigenic gene of Eimeria tenella ligated with the chicken cytokine IFN-γ、IL-18into the vector pcDNA4.0 to construct immuno-regulative DNA vaccinepcDNA4.0-MZ5-7-IFN-γ、pcDNA4.0-MZ5-7-IL-18. The protective effects the abovementioned DNA vaccine together with pcDNA4.0-MZ5-7、pcDNA4.0-MZ5-7-IL-2、pcDNA4.0- MZ5-7-IL-17 were tested in the protective experiment of chickens against thechallenge of Eimeria tenella
IPTG was used to induce the expression of pET28b-MZ5-7 constructed in ourlabouratory. Purified MZ5-7 protein was used to immune rats for E. tenelIa MZ5-7anti-serum. The anti-serum was used to check the expression of MZ5-7 DNA vaccine inchicken by western-blot. Designed special primers were used to check the transcription ofMZ5-7 DNA vaccine in chicken by RT-PCR. The results showed that the DNA vaccinescould be well transcripted and expressed in chicken and they could be used to animalprotective experiment.
The chicken of protective experiment were divided into eight groups: pcDNA4.0、pcDNA4.0-MZ5-7、pcDNA4.0-MZ5-7-IL-18、pcDNA4.0-MZ5-7-IL-17、pcDNA4.0-MZ5-7-IL-2、pcDNA4.0-MZ5-7-IFN-γ、Unimmunized-unchallenged controlgroup、unimmunized-challenged control group. Each group had 30 chicken and were injected with vaccine through chest muscle. The vaccine was inoculated at 14 days and 21days of age and each chicken was challenged with 5×10~4 Eimeria tenelIa sporulated oocytesat 28 days of age of chicken. All chicken were killed at 35 days of age and the protectiveeffects were estimated by the index of lesion score of caecum, OPG, average body weightgain, anticoccidia index (ACI), The results showed that the immuno-regulative DNAvaccine pcDNA4.0-MZ5-7-IL-18、pcDNA4.0-MZ5-7-IL-2 pcDNA4.0-MZ5-7-IFN-γhadgood protective effects. Separately their ACIs are 188.45、184.47、184.99.
引文
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