溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护研究

为研究溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护作用,实验构建了重组真核表达质粒pcDNA-flaC并将该质粒肌肉注射红笛鲷,采用PCR、RT-PCR、ELISA和攻毒试验等方法检测了该真核表达质粒在红笛鲷组织内的分布、表达和对红笛鲷的免疫保护.PCR结果显示,免疫接种7和28 d,注射点周围肌肉、鳃、肾脏、肝脏和脾脏都存在质粒分布；RT-PCR结果显示,免疫接种后第7天、14天和28天,红笛鲷不同组织内均有目的基因表达.ELISA结果表明,鱼血清内产生了抗FlaC蛋白的抗体,表明DNA疫苗免疫后鱼体表达了目的蛋白,并诱导产生了相应抗体.攻毒实验表明,免疫后的红笛鲷能较好地抵抗致病性溶藻弧菌的感染.结果表明,质粒pcDNA-flaC可能是抵抗溶藻弧菌感染的有效的疫苗候选物.

Immunogenic and protective effects of a DNA vaccine containing flagellin flaC gene against Vibrio alginolyticus in red snapper(Lutjanus sanguineus)

In order to study the immunogenic and protective effects of DNA vaccine,plasmid DNA encoding flagellin flaC gene(designated as pcDNA-flaC)was used as a DNA vaccine to immunize red snapper(Lutjanus sanguineus).The distribution,expression and immunoprotection of the DNA vaccine were analyzed in tissues of the red snapper by PCR,RT-PCR and challenge test.PCR results indicated that pcDNA-flaC was distributed in liver,spleen,kidney,gill and injection site muscle at 7-28 days after vaccination.RT-PCR results indicated that the flaC gene was expressed in all above tissues of vaccinated fish at 7-28 days after vaccination.These results demonstrated that the DNA vaccine was distributed and flaC gene was expressed in various tissues of vaccinated fish.Red snapper immunized with DNA vaccine showed higher serum antibody levels at 7-28 days after vaccination,compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1 and PBS.In addition,fish immunized with DNA vaccine developed a protective response to live V.alginolyticus challenge 28 days post-inoculation,as demonstrated by increased survival of vaccinated fish over the control fish.This study indicates that pcDNA-flaC is an effective vaccine candidate against V.alginolyticus infection.