嗜水气单胞菌分泌蛋白重组疫苗对斑马鱼的免疫保护效力评价

Immune protective efficacy of recombinant vaccine against Aeromonas hydrophila secreted proteins on zebrafish

嗜水气单胞菌(Aeromonas hydrophila)是一种常见的水产致病菌,可引发人畜鱼共患疾病,对水产养殖业造成严重危害,因此预防其传播和感染是当前亟待解决的问题。为开发高效的潜在亚单位疫苗,并为防治嗜水气单胞菌病害提供理论依据,基于前期的研究结果,选取在嗜水气单胞菌强毒株LP-2中表达丰度较高的8种分泌蛋白(ORF0322、ORF3982、ORF2874、ORF1767、ORF3984、ORF2546、ORF0472、ORF1609)作为研究目标,首先利用实时荧光定量PCR(qPCR)技术对其进行基因表达检测,结果发现这8种分泌蛋白在细胞中均可正常表达;然后将这些蛋白进行基因克隆与表达纯化,并将纯化的重组蛋白免疫斑马鱼(Danio rerio),连续培养28 d后,发现斑马鱼中相关免疫基因均发生上调,说明这些分泌蛋白可引起宿主发生免疫反应;最后,对这8种分泌蛋白进行免疫保护评价,细菌攻毒实验结果显示,其中6种分泌蛋白(ORF3982、ORF2874、ORF1767、ORF3984、ORF0472、ORF1609)的相对免疫保护率(Relative immune protection rate,RPS)大于50%,可作为候选疫苗开发。

Aeromonas hydrophila is a prevalent aquatic pathogen that poses a risk to fishery production and can cause zoonotic diseases.Therefore,prevention of transmission and infection of A.hydrophila is a critical issue to address.To develop a poten-tial subunit vaccine and provide a theoretical basis for better preventing and treating A.hydrophila disease,we selected eight secreted proteins(ORF0322,ORF3982,ORF2874,ORF1767,ORF3984,ORF2546,ORF0472 and ORF1609)from an A.hydro-phila virulent strain LP-2 based on our previous research.The qPCR test shows that all selected proteins were expressed nor-mally,then they were cloned and purified.The cloned and purified recombinant proteins were administered to zebrafish through injection.After 28 days of immunization,qPCR analysis detects increasing expression of all relevant immune genes in the zebrafish,suggesting that these recombinant proteins were capable of eliciting an immune response.In order to evaluate the immune protection offered by the eight selected secreted proteins,a bacterial challenge experiment was conducted.The results show that six of the secreted proteins(ORF3982,ORF2874,ORF1767,ORF3984,ORF0472,ORF1609)exhibited a relative im-mune protection rate(RPS)greater than 50%,indicating that they held promise as vaccine candidates againstA.hydrophila infection.