【作者】 张娜；

【导师】 李祥瑞；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 鸡球虫病是危害养鸡业健康发展的重要寄生虫病之一,目前,鸡球虫病的防治仍主要依赖于化学药物,但是抗药性问题和药物残留而引起的动物源性食品安全问题,给养禽业带来的负面影响不容忽视,与传统疫苗相比,核酸疫苗具有可靠的安全性、高稳定性、能诱导机体产生全面的免疫应答等诸多优势。NA4基因是E.necatrix子孢子表面抗原基因序列,其重组蛋白对E.necatrix经口感染雏鸡能产生部分保护力。IFN-γ、IL-2主要由活化T细胞产生,有着广泛的免疫调节作用,在鸡球虫保护性免疫中起着重要作用。本研究将毒害艾美耳球虫NA4基因单独或与鸡IFN-γ、IL-2基因融合克隆入pVAX1.0制备免疫调节型DNA疫苗,并用构建的DNA疫苗免疫动物,检测对E.necatrix攻虫的保护作用。用柔嫩艾美耳球虫子孢子表面抗原TA4基因特异性引物,以纯化E.necatrix孢子化卵囊总RNA为模板,RT-PCR方法克隆出了一段新基因序列。该基因全长747bp,共编码248个氨基酸,与国外Brothers从E.necatrix孢子化卵囊表面蛋白纯化而得NA4基因同源性达到99.7%,与柔嫩艾美耳球虫TA4基因同源性达到91.4%,拥有与柔嫩艾美耳球虫TA4抗原基因相同序列的一段信号肽,推测该基因能够诱导鸡产生同时抗毒害艾美耳球虫和柔嫩艾美耳球虫的保护性免疫反应。此基因已登录GenBank,登录号为EU523548。将NA4基因克隆至质粒表达载体pET-28a（+）,成功构建了重组表达质粒pET-28a（+）-NA4。用CaCl₂法将其转化至宿主细菌E.Coli BL21,并用IPTG成功诱导了NA4重组抗原在大肠杆菌表达。经SDS-PAGE分析其相对分子质量约为26 000KD。利用DNA重组技术,分别构建pVAX1.0-NA4、pVAX1.0-NA4-IFN-γ、pVAX1.0-NA4-IL-2 DNA疫苗。酶切鉴定正确后,分别用重组质粒免疫14日龄鸡,1周后取注射部位、非注射部位肌肉组织,用RT-PCR,Western-blot方法检测保护性抗原的表达情况。结果表明NA4基因、IFN-γ、IL-2基因均能在注射部位肌肉里成功表达。NA4重组蛋白、重组质粒pVAX1.0-NA4、pVAX1.0-NA4-IFN-γ、pVAX1.0-NA4-IL-2经腿部肌肉分别于14、21日龄两次免疫雏鸡,并设立感染非免疫和非感染非免疫对照。28日龄经口接种新鲜的E.necatrix孢子化卵囊,一周后宰杀全部实验动物,进行小肠病变计分、盲肠内容物卵囊数、增重和抗球虫指数四个指标进行统计,检测其保护力的大小。结果表明,所构建的免疫调节型DNA疫苗对感染球虫鸡具有良好的保护效果,其中以pVAX1.0-NA4-IL-2重组质粒组保护效果最好,有可能作为侯选的毒害艾美耳球虫和柔嫩艾美耳球虫DNA疫苗。更多还原

【Abstract】 The coccocidiosis of chicken is a cytoplastic entozoic parasite disease and may cause severe economic loss to the industry of poultry.Current methods for coccidiosis control in the poultry industry have many drawbacks,such as drug-resistance and the potential reversion to virulence.Therefore,DNA vaccines containing important parasitic antigens have been suggested as an alternative to the virulent or attenuated vaccines.The NA4 antigen is sporozsoite surface antigen of E.necatrix,and has been shown to elicit partial protection in chicken against an oral challenge with E.necatrix.Chicken IFN-γand IL-2 are cytokines which play an important role in immune regulatory network.In this report,we constructed immune regulative DNA vaccine by the combination of NA4 gene of E.necatrix with chicken IFN-γand IL-2 and tested their protective effects on the chicken against the challenge of E.necatrix.By means of rectum inoculating merozoite of E.necatrix to purify E.necatrix oocysts,use specific primer of TA4 gene of sporozsoite surface antigen of E.tenella,a new gene was cloned with RT-PCR from the sporulated oocysts of E.necatrix,the new gene is 747bp and encodes 248 amino acids.Comparison of the NA4 with the nucleotide sequence of Brothers screemed from surface protein of E.necatrix oocysts,they have the sequence homologies of 99.4%,and comparison of NA4 to TA4 gene of sporozsoite surface antigen of E.tenella suggests that they have the sequence homologies of 91.4%and have a gene encodes a 23 amino acid signal peptide.We can suppose that NA4 antigen can induce protective immune reaction to E.necatrix and E.tenella.The sequence has been seperated in GenBank.The NA4 gene was then cloned into pET-28a(+) vector to form a prokaryotic expression plasmid.IPTG induced expression of NA4 gene in E.coli BL21 induced a specific 26kD protein band in SDS-PAGE of bacteria proteins. By the means of DNA recombination technique,pVAX1.0-NA4, pVAX1.0-NA4-IFN-γand pVAX1.0-NA4-IL-2 vectors were constructed.After identified by restriction enzyme digestion,these recombinant DNA plasmids were extracted and injected into the leg muscle of 14 days old chicken.A week later the injected tissue was sampled respectively to check whether these DNA plasmid expressed or not by RT-PCR and Western-blotting.The results indicated that NA4 gene of E.necatrix and IFN-γ,IL-2 gene of chicken could be successfully transcribed and expressed in the injected tissues.pET-28a(+)-NA4 recombinant protein and pVAX1.0-NA4、pVAX1.0-NA4-IFN-γ、pVAX1.0-NA4-IL-2 recombinant DNA plasmid were inoculated respectively into the leg muscle of chicken twice at 14 and 21 day-old of age,and the control groups were also set up,then challenged with E.necatrix sporulated oocysts at 28 days old.The indexs of weight gain,oocysts per gramfeces(OPG),reduction of lesion scores and anti-coccidial index(ACI) were done respectively to check the protection of these DNA vaccines against E.necatrix infection.The results indicated that the immune-regulating DNA vaccines could well protect chicken from E.necatrix infection.The best one of them was pVAX1.0-NA4-IL-2 recombinant DNA vaccine,so it could be a satisfying candidate nucleic acid vaccine against E.necatrix and E.tenella.更多还原