传染性造血器官坏死病毒表面糖蛋白基因核酸疫苗的构建及对虹鳟血液生化指标的影响

Construction of glycoprotein gene nucleic acid vaccine against infectious hematopoietic necrosis, and its effect on serum biochemical indices

为研究传染性造血器官坏死病毒(infectious hematopoietic necrosis virus, IHNV)表面糖蛋白(glycoprotein, G)基因核酸疫苗对虹鳟免疫保护效果及血液生化指标的影响,将IHNV G基因克隆至pMD19-T载体中,连接产物在DH5α中进行转化,获取重组质粒pMD19-TG后回收G基因片段。将鉴定正确的G基因片段利用Bam H I和Xho I酶切位点克隆在真核表达载体pVAX1上,构建核酸疫苗pVAX1-G。重组质粒pVAX1-G按照8μg/尾的剂量注射虹鳟设为pVAX1-G组,同时设8μg/尾空质粒组、PBS对照组和空白组,于免疫后21 d,以100倍半数组织培养感染剂量(tissue culture infective dose, TCID50)通过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),攻毒后收集免疫虹鳟血清进行血液指标检测。结果显示,攻毒后虹鳟血清中16项指标中谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、总胆红素(TBIL)、总胆汁酸(TBA)、葡萄糖(GLU)、尿素(Urea)、肌酐(CREA)、总蛋白(TP)、白蛋白(ALB)和球蛋白(GLO)与正常虹鳟相比有显著变化,空载组11项指标较pVAX1-G组变化显著;攻毒后14 d pVAX1-G组累积死亡率为19%(19/100),而空载组和PBS对照组分别为62%(62/100)和85%(85/100)。pVAX1-G核酸疫苗对虹鳟免疫保护率为78%。病理学观察发现,免疫pVAX1-G组虹鳟的肝脏、脾脏、肾脏组织未见明显损伤。综上表明,pVAX1-G作为核酸疫苗有助于减轻IHNV对虹鳟的损伤,对IHNV有较好的免疫保护效果。

The aim of this study was to study the effect of infectious hematopoietic necrosis virus(IHNV) nucleic acid vaccine on the immune protection of Oncorhynchus mykiss and serum biochemical indices of Oncorhynchus mykiss. Cloning the glycoprotein gene(G) into pMD19-T vector, we transformed the linked product in DH5α,obtaining recombinant plasmid pMD19-T-G, and recovering the G gene fragment. The identified correct G gene fragment was cloned on eukaryotic expression vector pVAX1 using Bam H I and Xho I cleavage sites to construct nucleic acid vaccine pVAX1-G. The recombinant plasmid pVAX1-G was injected into O. mykiss at a dose of 8μg/ind. as pVAX1-G group, while 8 μg/ind. empty group, PBS control group and blank group were set up.Twenty-one days after immunization, an anti-virus experiment was carried out. After anti-virus, serum was collected and blood indexes were tested. The results showed that there were significant changes in 16 indexes in O.mykiss serum after the attack: glutamic-pyruvic transaminase(ALT), glutamate transaminase(AST), alkaline phosphatase(ALP), total bilirubin(TBIL), total bile acid(TBA), glucose(GLU), the urea(Urea), creatinine(CREA), total protein(TP), albumin(ALB) and globulin(GLO) were compared with normal O. mykiss, and the changes of 11 indexes in no-load group were more significant than those in pVAX1-G group. The cumulative death rate of pVAX1-G group was 19%(19/100)14 days after the attack, while that of no-load group and PBS control group was 62%(62/100) and 85%(85/100), respectively. The protective rate of pVAX1-G nucleic acid vaccine for O. mykiss was 78%. The results showed that pVAX1-G as a nucleic acid vaccine was helpful to reduce IHNV’s injury to O. mykiss and had better immune protection effect against IHNV.