| 摘要: |
| 利用DNA重组技术，将柔嫩艾美耳球虫（Eimeria tenalla）第2代裂殖子抗原基因MZ5-7的完整阅读框（ORF，945 bp）克隆到真核表达质粒pcDNA4.0中，构建了DNA疫苗pcDNA4.0-MZ。用同样方法，将鸡成熟白细胞介素-17（IL-17）基因的完整阅读框（507 bp）与MZ5-7基因串连，使MZ5-7基因位于IL-17基因的上游，将串连基因克隆到pcDNA4.0中，构建成免疫调节型DNA疫苗pcDNA4.0-MZ-IL-17。2种DNA疫苗纯化后，胸部肌肉注射免疫1周龄雏鸡（50 μg/鸡），在免疫后3，5，7，15和20 d取注射和非注射部位肌肉组织，分别用RT-PCR和Western blot方法检测抗原基因转录和表达情况。结果表明，在2种疫苗免疫后5，7和15 d， 用RT-PCR方法在注射部位均可检测到MZ5-7基因的转录产物，大小约950 bp，但在免疫第3天和20天的肌肉组织中未检测到表达产物。相同时间用Western blot方法进行检测，结果在免疫5和7 d的鸡肌肉组织中检测到表达产物，pcDNA4.0-MZ表达产物大小约36 ku，与理论推测值一致，pcDNA4.0-MZ-IL-17表达产物大小约64 ku，较MZ5-7和IL-17基因之和的理论推测值58 ku略大。但在非注射部位，两种方法均未检测到转录或表达产物。上述结果表明，通过肌肉注射后，两种DNA疫苗均可在鸡肌肉组织中得到有效表达。 |
| 关键词: 柔嫩艾美耳球虫 MZ5-7基因 IL-17 DNA疫苗 鸡 |
| DOI： |
| 分类号: |
| 基金项目:国家“863”计划项目“禽用抗球虫基因工程疫苗的研制”（2002AA245061） |
|
| Constructions of two DNA vaccines against chicken E.tenella and their expressions in chicken muscles |
|
|
| Abstract: |
| The open reading frame of MZ5-7 gene,an antigen of Eimeria tenella second generation merozoites，was cloned into plasmid pcDNA4.0 to construct DNA vaccine pcDNA4.0-MZ.With the same methods,the mature interlukin 17 gene of chicken was cloned into downstream of the MZ5-7 gene to construct an immune regulatory DNA vaccine pcDNA4.0-MZ-IL-17 co-expressive of MZ5-7 antigen and IL-17.After being purified,50 μg of these two DNA vaccines were used to immunize one-week-old chickens with breast muscle injection respectively.The transcriptions and expressions of the target genes were determined by RT-PCR and Western-blot respectively on the day 3,5,7,15 and 20 post immunization.The results showed that the transcripted products of MZ5-7 gene was detectable from all injection site muscles immunized with two DNA vaccines respectively on day 5,7 and 15 post immunization using RT-PCR.The expression proteins of two plasmids were respectively identified by Western-blot from all injection sites on day 5 and 7 post immunization.The molecular weight of expressive protein of pcDNA4.0-MZ was 36 ku coincident with the predicted protein of the MZ5-7 gene while the molecular weight of expressive product of pcDNA-4.0-MZ-IL-17 was about 64 ku,slightly larger than that of predicted product of MZ5-7 gene plus IL-17 gene.No target products were detected in the no-injection sites immunized with any of the two DNA vaccines.The results indicated that these two DNA vaccines could be effectively expressed in chicken muscles when immunized by muscle injection. |
| Key words: Eimeria tenella MZ5-7 gene IL-17 DNA vaccine chicken |
