Construction Of Recombinant Lactococcus Lactis Expressing VP1 Protein Of Duck Hepatitis A Virus Type 1 And Evaluation Of Immune Effect

Duck Viral Hepatitis(DVH)is caused by Duck Hepatitis Virus(DHV).Ducklings under 3 weeks of age are mostly infected.The diseased ducks are characterized by neurological symptoms and liver disease.It is an acute,highly lethal infectious disease.DHV mainly includes Duck Hepatitis A Virus(DHAV)and Duck Astrovirus(DAstV).DHAV is divided into DHAV-1,DHAV-2,and DHAV-3 according to the differences in serotypes.At present,DHAV-1 is more virulent,and the infection situation is becoming more and more complicated.Generally,other diseases cause mixed infections,which seriously harms Development of the duck industry.In the actual production,DHAV-1 vaccine is seldom used to prevent and control DHAV-1,and yolk antibody is injected into ducklings after DHAV-1 infection,and the effect is better.Based on the characteristics of beneficial bacteria,lactobacillus has been widely used in food fermentation,drug production,feed addition and other fields.In recent years,lactobacillus has begun to be applied in the fields of molecular genetics and enetic engineering.In particular,the potential of lactobacillus to have oral vaccine with recombinant protein carrier has attracted much attention.Based on the characteristics of beneficial bacteria,lactic acid bacteria(LAB)has been widely used in food fermentation,drug production,feed addition and other fields.In recent years,it has begun to be used in molecular genetics and genetic engineering.Especially,the potential of LAB as a recombinant vaccine for oral vaccine has attracted much attention.In order to reduce the use of antibiotics in duck farming,reduce the risk of susceptibility,reduce the mortality of large groups,avoid the risk of virulence recovery and immune failure,and enable ducklings to obtain protective antibodies during the susceptible period,The purpose of this study is to develop a new type of microecological preparation,which can make full use of molecular biology technology to modify lactobacillus on the level of genetic engineering.This study constructed the major capsid protein VP1 of DHAV-1 into the Lactococcus lactis expression system that has been studied,and used the transcript and translation level elements of the gene to make use of Lactococcus lactis for living.The carrier presents the antigen protein to the outside of the cell,stimulates the body's mucosal immune system,generates specific antibodies,and protects the body against the attack of DHAV-1.The research content mainly includes the following two parts:Part 1: Construction of Recombinant Lactococcus lactis of DHAV-1 VP1 ProteinIn order to secrete the exogenous protein expressed intracellularly to the outside of the cell in time,It was selected Lactococcus lactis milk fat subspecies MG1363 as the carrier and used the constitutive E.coli-lactic acid bacteria shuttle plasmid pMG36 e with rolling circle replication as the expression vector.The target protein is further promoted to achieve extracellular secretion expression.A Usp45 signal peptide sequence was fused to the Nterminus of VP1,and the target protein is transported out of the cell wall across the membrane under the guidance of the signal peptide,and the mature protein was released outside the cell.Fusion PCR was used to construct the plasmid Usp45-VP1-pMG36 e.The recombinant plasmid was transformed into Lactococcus lactis MG1363 by electroporation,and Usp45-VP1-pMG36 e / MG1363 recombinant Lactococcus lactis was successfully constructed.The intracellular and extracellular expression of VP1 protein in recombinant lactococcus lactis was identified,and the Western Blot test results proved that specific VP1 protein was detected both inside and outside the bacterial body.The fusion protein VP1 with a size of about 26 kDa was expressed.At the same time,indirect immunofluorescence experiments were performed on the recombinant lactic acid bacteria.The VP1 recombinant lactic acid bacteria gave green fluorescence under FITC fluorescent labeling.Part 2: Evaluation of the immune effect of VP1 protein recombinant LABRecombinant Lactococcus lactis MG1363 and pMG36 e empty vector plasmid recombinant Lactococcus lactis MG1363 containing Usp45-VP1-pMG36 e plasmid were orally immunized with one-day-old ducklings for 7 days,and the blank group was used as a negative control.ELISA detected the levels of DHAV-1 specific antibodies and cytokines in serum and small intestinal fluid 24 hours after oral immunization,and evaluated the immune effect.The results showed that the specific IgG antibodies and intestinal mucosal SIgA antibodies in the serum of the experimental group of VP1 recombinant lactococcus.The levels of IL-2,IL-4,IL-10,and IFN-? were significantly increased compared with other control groups(P <0.05).On the eighth day,two groups of ducklings injected with DHAV-1 virulent strain were assigned to each group to simulate natural infection.The mortality of each group of ducklings was monitored.In the end,the protection rate of ducklings in the VP1 recombinant LAB group was 80%.The LAB live vector vaccine constructed in this study had been provided a new way for the prevention and control of DHAV-1 in the future.