Construction Of DNA Vaccine And Its Protective Efficiency On The Largemouth Bass(Micropterus Salmoides) Challenged By Largemouth Bass Virus(LMBV)

Largemouth bass is one of the most important economic fishes for freshwater aquaculture in China.In recent years,the demanded of largemouth bass in China has been increasing,which has led to an increase in the breeding scale and breeding density of domestic largemouth bass.The outbreak of diseases of largemouth bass,especially viral diseases,is also more frequent.Largemouth Bass Virus(LMBV),a species of the genus Ranavirus in the family iridoviridae,can cause explosive death,making significant economic losses.At present,the research on LMBV virus mostly focus on the molecular mechanism and physiological level,but the development of LMBV vaccine has not been reported yet.Therefore,in order to prevent viral disasters caused by LMBV,this study constructed a recombinant plasmid p CDNA3.1(+)-MCP expressing LMBV major capsid protein(MCP)as DNA vaccine.First,the expression of recombinant plasmids in fish cells was detected from m RNA and protein levels by PCR,RT-PCR and Western Blot.Then,through hematological analysis,serum neutralizing antibody titer analysis,expression analysis of immune-related genes,and distribution of plasmids in fish,the immune level of the vaccinated largemouth bass was detected.Finally,the largemouth bass was challenged by LMBV and then analyzed the relative percentage survival rate(RPS).The result showed that the recombinant plasmid could correctly transcript and express the target protein in fish cells and still expressed for 96 h in the EPC cells.The RPS of the DNA vaccine group was 62.5% in the challenge experiment,which was significantly higher than the 23.1% in the empty group and 0% in the PBS group.In addition,on the 1 st,7 th,14 th,21 st and 28 th day post vaccination(dpv),the blood was collected and the liver,spleen and head kidney tissues were extracted.The results showed that p CNDA3.1(+)-MCP could be detected in liver,spleen and head kidney tissues,but the expression level was higher on the 7 dpv and 14 dpv.For the DNA vaccine group,the number of red blood cells were significantly increased at 14 dpv and 21 dpv,moreover,the proportion of neutrophils and monocytes in the leukocytes were also remarkably increased but the proportion of lymphocytes was decreased.The result of serum neutralization titer demonstrated that antibodys corresponded to recombinant plasmid were existing in serum of largemouth bass and reaching the peak at 14 dpv(the titer was 1:(375.00±40.26)),significantly higher than the control group.The q RT-PCR results showed that the m RNA expression levels of IL-8,IL-1?,TNF-? and Mx genes in the liver,spleen and head kidney tissues were significantly up-regulated at the vaccine group.The expression level gradually decreased as the immunization time prolonged.In summary,the recombinant plasmid expressing the LMBV MCP protein can induce a significant immune response in the largemouth bass,and this DNA vaccine can be used as a potential vaccine against the LMBV virus.It provides a theoretical basis for the production and industrialization of LMBV DNA vaccine.