小瓜虫抑动蛋白基因核酸疫苗制备

DNA vaccine preparation ofichthyophthirius immobilization protein

为防治水产养殖动物常见多发的小瓜虫病,本试验选取小瓜虫抑动蛋白基因,人工合成经物种优化后的抑动蛋白基因序列,以pVAX1为载体构建重组质粒,转入大肠杆菌DE3进行克隆表达。大量提取构建的重组质粒,通过肌肉注射免疫鱼体,注射剂量为125μg/尾,同时设立空载体对照组及空白对照组。首免后第4周进行二次加强免疫,分别于首免后第2、3、4周,二次加强免疫后第1、2、3、4、8、16周,抽样采集各组血液,分离血清,RT-PCR法检测抑动蛋白转录水平,同时检测血清抗体产生情况。二次加强免疫后第2周进行同居感染及人工感染试验,检测免疫保护效果。结果表明,质粒注射入鱼体后,所有抽查血液样本均检测到目的基因的转录;所有抽查免疫鱼血清样本均可以对小瓜虫幼虫产生制动效果,制动时间长达8周,空载体对照组和空白对照组无制动效果;同居感染及人工感染后,核酸疫苗免疫组存活率达到90%以上,而空载体组存活率为20%~40%,空白对照组全部死亡。表明构建的质粒作为核酸疫苗对小瓜虫病有较好的免疫保护效果,可显著降低死亡率。研究结果为小瓜虫核酸疫苗的研发与生产应用奠定了基础。

In order to combat ichthyophthiriasis in aquaculture animals,immobilization was selected as the protein gene to recombinant plasmid vector with the pVAX1 as the carrier,transformed into E.coli DE3 for cloning expression,then extracted the recombinant plasmids,intramuscular immunizaed fish as a dose of 125μg/tail,while established the empty vector control group and the control group and strengthen the immune after 4weeks.Collect each group blood samples at 2,3,4weeks after the first immunization and 1,2,3,4,8,16 weeks after the second immunization respectively,Immobilization protein transcript levels and the serum antibody generation were detected.At the same time,2weeks after the second strengthen immune cohabitation infection experiment and artificial infection test were carried out to detected the immune protection.The results showed that in the fish injected plasmid,all sample blood samples detected target gene transcription and could inhibit ichthyophthiriasis activity for 8weeks.After cohabitation infection and artificial infection test,the survival rate of DNA vaccine group was up to 90%,while the empty vector group was 20%-40% and the control group all dead,which confirmed that the DNA vaccine for ichthyophthirius has better immune protection.It laid a foundation for the production and applications of the DNA vaccine of ichthyophthirius.