捻转血矛线虫DNA疫苗的构建及山羊免疫保护性试验.pdfVIP

中国农业科学 2007,40(12):2869-2875 Scientia Agricultura Sinica 捻转血矛线虫DNA疫苗的构建及山羊免疫保护性试验 严若峰，徐立新，孙延鸣，赵光伟，李祥瑞 （南京农业大学动物医学院，南京 210095 ） 摘要：【目的】构建捻转血矛线虫DNA疫苗，并研究该疫苗对山羊的免疫保护效果。【方法】将捻转血矛线虫 重要保护性抗原H11的编码基因分H11-1、H11-2和H11-3 三部分，分别亚克隆到基因疫苗载体pcDNA4/His Max C 中，用酶切反应、序列测定等方法鉴定重组疫苗。将纯化的DNA疫苗肌肉注射山羊后，用RT-PCR、Western blotting 和 ELISA 方法检测疫苗在山羊体内的转录、翻译和诱导 IgG 的产生。第 2 次免疫后 2W 用 10 000 条捻转血矛线虫 第3期幼虫攻击实验动物，检测山羊虫卵排出、虫卵孵化率、成虫数量等免疫保护性指标。【结果】获得了捻转血 矛线虫H11 DNA疫苗，疫苗第1次免疫7d 后在山羊肌肉组织中进行了转录，第2次免疫7 d后，DNA疫苗获得了 翻译，并诱导机体产生了相应的抗体。与对照组比较，试验组山羊排出虫卵减少58.8%、虫卵孵化率减少75.3%、 成虫减少68.2%。【结论】构建了捻转血矛线虫H11 DNA疫苗，该DNA疫苗对山羊具有较好的免疫保护效果。 关键词：山羊；捻转血矛线虫；H11；DNA疫苗 Construction of DNA Vaccines for Haemonchus contortus and Its Protective Effects in Goats YAN Ruo-feng, XU Li-xin, SUN Yan-ming, ZHAO Guang-wei, LI Xiang-rui (College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095) Abstract: 【Objective 】The study was carried out to construct DNA vaccines for Haemonchus contortus, and test the protective effects in goats. 【Method 】Three gene fragments encoding parts of H. contortus H11, H11-1, H11-2 and H11-3, were inserted into pcDNA4/His Max C. The recombinants were identified by double restriction enzyme digestion and sequence analysis. Lambs were immunized with DNA vaccines about 28 and 14 days before challenged by 10 000 H. contortus L3. The expression of vaccines in goats was analyzed by RT-PCR and Western blotting. While the IgG stimulated with the vaccines was checked by ELISA. After challenge with L3, EPG (eggs per gram feces), L3 hatched from eggs and worm burdens of lambs were counted. 【Result 】 Transcriptions and translations of the vaccines in goat muscles were confirmed at se