【作者】 阎雪飞；

【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不错的应用前景。为此我选择球虫的抗原NPmz19基因与鸡细胞因子IFN-γ、IL-2基因,通过DNA重组技术分别构建了真核重组质粒pVAX1.0-NPmz19,pVAX1.0-NPmz19-IFN-γ、pVAX1.0-NPmz19-IL-2,进行动物免疫保护性试验比较这些真核重组质粒的免疫保护效果。根据GenBank上报道的NPmz19基因序列,利用计算机设计一对引物,从收集的E.necatrix裂殖子中提取总RNA,然后应用RT-PCR技术扩增出E.necatrix NPmz19基因并对其进行测序。测序结果表明,基因全长960bP,比GenBank上报道的NPmz19基因序列少六个核苷酸,具有一个完整的开放阅读框,编码319个氛基酸,所得序列已提交到GenBank,登录号为EU795423,与已知序列相比较,核苷酸和氨基酸同源性分别为94%和88%,51个碱基发生变异,24个氨基酸发生变异,但没有变异成终止密码子,NPmz19基因编码约35KD的蛋白。将上述克隆得到的NPmz19基因克隆到原核表达载体pET32a(+)中,构建重组质粒pET32a(+)-NPmz19,并转化到大肠杆菌E.coli BL21中,通过IPTG诱导表达重组NPmz19基因片段编码的蛋白。SDS-PAGE电泳显示,表达的重组蛋白分子量在35KD左右,且在诱导4-5小时后表达量最多。细菌经超声波裂解破碎,1%TritonX-100洗涤后,SDS-PAGE电泳显示,pET32a(+)-NPmz19的表达产物绝大部分存在于包涵体中。包涵体经过初步分离纯化、变复性,再经紫外分光光度计检测,结果表明,纯化的重组蛋白浓度为4.6mg/mL。利用DNA重组技术,分别构建pVAX1.0- NPmz19,pVAX1.0-NPmz19-IFN-γ、pVAX1.0.NPmz19-IL-2真核重组质粒。酶切鉴定正确后,分别用重组质粒免疫14日龄鸡,1周后取注射部位肌肉组织,用RT-PCR,Western blot检测抗原的表达情况。结果表明该NPmz19基因和细胞因子均能在注射部位肌肉中表达。NPmz19重组蛋白、重组质粒pVAX1.0-NPmz19,pVAX1.0-NPmz19-IFN-γ、pVAX1.0-NPmz19-IL-2经腿部肌肉注射分别于14、21日龄两次免疫小公雏。28日龄经口接种新鲜的E.necatrix孢子化卵囊,一周后宰杀,然后分别作小肠病变计分、测定克盲肠内容物卵囊数、增重、综合抗球虫指数(ACI)几个指标,确定其保护力的大小。结果表明,构建的毒害艾美耳球虫真核重组质粒能有效地降低克盲肠内容物卵囊数、减少小肠病变和提高相对增重。pVAX1.0-NPmz19、pVAX1.0-NPmz19-IFN-γ、pVAX1.0-NPmz19-IL-2、NPmz19重组蛋白的克盲肠内容物卵囊数平均值分别为1.04×10~5,0.93×10~5,0.65×10~5和1.56×10~5,小肠病变平均计分依次为1.03、0.97、0.69、1.53,相对增重率分别为91.18、93.78、96.61和85.56,综合抗球虫指数ACI分别是170.88、182.98、188.71、160.26。综合各项数据结果,发现pVAX1.0-NPmz19-IL-2真核质粒保护效果最佳,因此可以作为毒害艾美耳球虫的候选DNA疫苗。更多还原

【Abstract】 Avain coccocidiosis,an enteritis caused by Eimeria infection,is of major economic importance to the poultry industry around the world.The development of poultry industry was restricted by the emergence of drug-resistant parasites and the drug residues problem so that a new approach to prevent avain coccocidiosis should be found.Stimulating the cell immuno-reaction of the host has been approved to be an effective approach to prevent avain coccocidiosis.The DNA vaccine,as the third generation of vaccine,has the obvious advantage to induce abroad cell immuno-reaction and form perdured immune memory. Especially,the immune regulative DNA vaccine,constructed by special antigen together with cytokine gene,has more prospect in the anti-coccocidiosis practice.In this investigation pVAX1.0-NPmzl9,pVAX1.0-NPmz19-IFN-γ,pVAX1.0-NPmzl9-IL-2 were constructed by the gene recombinant technology with the antigen NPmzl9 of Eimeria necatrix and the chicken cytokine IFN-γ,IL-2.The protective effects of the above mentioned eukaryotic recombinant plasmids together with NPmz19 protein were tested in the protective experiment of chickens against the challenge of Eimeria necatrix.Based on NPmzl9 sequence published on GenBank,a pair of special primers were designed with computer software.NPmz19 gene was amplified by RT-PCR with the total RNA extracted from E.necatrix merozoites as template.In order to identify it,the nucleotides were sequenced and compared with the existing data published on GenBank. The result indicated this NPmz19 gene encoded 960 nucleotides,319 amino acids residues, with a whole open reading frame,6 nucleotides lesser than the NPmz19 on Gen_Bank,the sequence has submitted to GenBank,the accession number is EU795423,and it had a 94% nucleotide homology and 88%amino acid homology with NPmz19 on GenBank,51 nucleotides and 24 amino acids are mutated.The NPmz19 gene encoded a 35kDa protein.NPmz19 gene was cloned into pET32a(+) vector,then transformed into E.coIi BL21. After induced by IPTG,SDS-PAGE indicated that about 35kDa recombinant protein was expressed.It was found that the target protein expressed to peak on the 4th and 5th hour after induction.To purify the recombinant protein,the inclusion body was isolated and washed by 1%Triton-X100.Then it was denatured by urea and renatured by dialyzation in PBS.Finally,the concentration of recombinant protein was 4.6mg/mL.With the DNA recombinant technique,the recombinant plasmids pVAX1.0-NPmz19, pVAX1.0-NPmz19-IFN-γ,pVAX1.0-NPmz19-IL-2 were constructed.After the restricted enzyme digestion,these three recombinant plasmids were then injected into the leg muscles of 14-day old chicken respectively,the injected muscles were analyzed by RT-PCR and western blot to confirm the expression of the antigen NPmz19 and cytokine gene.The result indicated that they can successfully expresse in chicken’s muscles.pET32a(+)-NPmz19 recombinant protein and pVAX1.0-NPmz19,pVAX1.0-NPmz19 -IFN-γ,pVAX1.0-NPmz19-IL-2 eukaryotic recombinant plasmids were injected into chicken’s leg muscles at the age of 14 and 21 day respectively.Chicken were then challenged with E.necatrix sporulated oocysts at 28 days.In order to evaluate the protective effects of these eukaryotic recombinant plasmids against E.necatrix,the parameters including weight gain,the oocysts per gram of caecum of caecum,the small intestine lesion score and anti-coccidial index(ACI) were observed.The Results indicated that these recombinate protein and eukaryotic recombinant plasmids were effective since they could reduce the relative oocyst production increase relative weight gain,and significantly reduce the small intestine lesion score.The small intestine lesion score of pVAX1.0NPmz19,pVAX1.0NPmz19-IFN-γ,pVAX1.0NPmz19-IL-2 and pET32a(+) -NPmzl9 were respectively 1.03,0.97,0.69,1.53,1.04×10~5,the counting number of caecum were respectively 1.04×10~5,0.93×10~5,0.65×10~5and 1.56×10~5 and the relative weight gain were respectively 91.18%,93.78%,96.61%and 85.56%,and ACI were respectively170.88,182.98,188.71,160.26.As reported above,pVAX1.0NPmz19-IL-2 is the best one of the three eukaryotic expression vectors,therefore it is promising to protect avian against E.necatrix.更多还原