传染性造血器官坏死病核酸疫苗的构建及其在虹鳟接种部位的消长规律

本研究将传染性造血器官坏死病病毒(infectious hematopoietic necrosis virus,IHNV)分离株SD-12糖蛋白(glycoprotein,G)基因克隆进商业化载体pc DNA3.1(+),构建了IHNV G的表达载体,即传染性造血器官坏死病(infectious hematopoietic necrosis,IHN)核酸疫苗,命名为p IHNsd-G。采用背鳍基部肌肉注射的方式,以2μg/尾的剂量免疫虹鳟(Oncorhynchus mykiss)鱼苗(5.0±0.5)g。于免疫后第4天及第7天,利用real-time PCR技术检测免疫虹鳟头肾及接种部位肌肉组织Mx-1基因表达情况;于免疫后第21天,以100倍半数组织培养感染剂量(tissue culture infective dose,TCID50)采取腹腔注射的方式进行攻毒实验,计算核酸疫苗相对保护率(relative percent survival,RPS);于免疫后第60天及150天检测免疫虹鳟血清IHNV中和抗体效价;最后,以p IHNsd-G的启动子序列和氨苄青霉素抗性基因序列为目标基因,利用PCR方法监测p IHNsd-G在免疫虹鳟接种部位的动态分布情况。结果显示:Mx-1基因在头肾和接种部位肌肉中均显著上调表达,并且在接种部位肌肉组织中明显高于同一时间点的头肾组织;攻毒实验中p IHNsd-G对虹鳟的相对保护率高达94.4%;而在免疫后第60天,所有免疫虹鳟血清中均存在中和抗体,其最高效价高达320,在免疫后第150天,最高抗体效价为80,自此,说明已成功获得有效的IHN核酸疫苗。p IHNsd-G在虹鳟接种部位的PCR监测结果显示:在免疫后的第1天即可在注射部位的肌肉中检测到全部p IHNsd-G目标片段,在第84天时已经无法从注射部位肌肉中扩增出全长氨苄青霉素抗性基因,而所有目标基因在第150天时均消失不见。本研究在成功构建IHN核酸疫苗并系统地验证了其有效性的基础上,开展了该疫苗在接种部位的动态分析研究,为IHN核酸疫苗的研发和安全性评价研究提供了基础数据。

Construction and the growth and decline pattern of an infectious hematopoietic necrosis nucleic acid vaccine in rainbow trout

In this study, the infectious hematopoietic necrosis virus (IHNV) isolate SD-12 glycoprotein (glycopro-tein, G) gene was cloned into commercial vector pcDNA3.1 (+) to construct an IHNV G expression vector, known as an infectious hematopoietic necrosis (IHN) nucleic acid vaccine and named pIHNsd-G. Rainbow trout (5±0.5) g were immunized with 2 μg of the vaccine by intramuscular injection at the dorsal fin base. The expression of the Mx-1 gene was detected by real-time PCR in the anterior kidney and the muscles from vaccine-delivered sites of the rainbow trout at 4 days and 7 days after immunization, respectively. At 21 days post immunization, the relative protection rate (RPS) of the vaccine was calculated by challenge with SD-12 at a dose of 100% of the tissue cul-ture-infective dose (TCID50) by intraperitoneal injection. The serum neutralizing antibody titers of immunized rainbow trout were tested at 60 and 150 days post-immunization. Finally, the pIHNsd-G promoter sequence and ampicillin resistance gene sequence were used as target genes and the dynamic distribution of pIHNsd-G in the inoculated part of the rainbow trout was monitored by PCR. The results showed that the Mx-1 gene was signifi-cantly up-regulated in the kidney and the muscles at the inoculated site and was significantly higher in the muscle tissue than in the head kidney at the same time point. Challenge of rainbow trout showed that the RPS of pIHNsd-G for rainbow trout was 94.4%. The neutralizing antibody titer revealed that at 60 days post-immunization, neutralizing antibodies were found in all of the serum samples of immunized rainbow trout and the highest titer was 320, while the highest antibody titer at 150 days post-immunization was 80. Since then, it has been shown that an effective IHN nucleic acid vaccine has been successfully obtained. The results of PCR following the injection of pIHNsd-G rainbow trout showed that all pIHNsd-G target fragments were detected in the muscle of the injection site on the first day after immunization; it was not possible to amplify the full-length ampicillin resistance gene from the injection site muscle at 84 days, and all target genes disappeared at 150 days. Based on the successful construction of an IHN nucleic acid vaccine and the systematic validation of its effec-tiveness, this study carried out dynamic analysis of the vaccine at the inoculation site, which provides basic data for the research and development of IHN nucleic acid vaccines and safety evaluations.