Construction Of Recombinant Lactococcus Lactis Expressing ?C Protein Of Novel Duck Reovirus And Evaluation Of Immune Effect

Novel duck reovirus(NDRV)disease is caused by a new duck derived virus different from Muscovy duck reovirus.the clinical of the disease is characterized by the liver showed irregular massive or patchy necrosis and spleen necrosis.Commonly known as "duck new liver disease","duck hemorrhagic-necrotic hepatitis".Recently,NDRV was commonly identified in various duck species,which has been becoming a threat to the duck-feeding industry of China.It has gradually attracted the attention of various scholars and experts.Vaccines are rarely used in production.Lactic acid bacteria(LAB)are common bacteria in human and animal intestines.It is recognized as a generally recoas safe microorganism.It is widely used in the fields of food,medicine,health products and feed addition.In recent years,the research on lactic acid bacteria has gradually deepened,and began to involve genetic engineering and genetics.LAB can also prepare oral vaccine,because of various advantages such as safety,non toxicity,convenient administration and good protective effect.Oral vaccine of LAB delivery antigen through gastrointestinal mucosa,induce animal body to produce efficient immune response.This study constructed the major capsid protein sigma C(?C)of NDRV into the Lactococcus lactis(L.lactis)expression system that has been studied,using LAB as carrier ?C protein is presented to the cell surface.Stimulate the body's mucosal immune system and produce specific antibodies.And protects the body against the attack of NDRV.The research content mainly includes the following two parts:Part 1: Construction of Recombinant L.lactis of NDRV ?C ProteinIn this experiment,L.lactis MG1363 was selected as the live bacterial vector,and the Escherichia coli-LAB constitutive shuttle plasmid p MG36 e was selected as the expression vector for the construction of LAB live vector vaccine expressing NDRV ?C protein.Firstly,the gene T7g10L-Usp45 and pgs A were fused by designing primers to obtain the T7g10L-Usp45-pgs A gene fragment,and then the plasmid T7g10L-Usp45-pgs A-?C-e GFP was constructed by homologous recombination technology,and the gene was inserted into the multi-cloning site region of the vector p MG36 e.Using Hind III and Xba I as insertion sites,a recombinant plasmid with a size of 6054 bp was constructed.The recombinant plasmid was transferred into L.lactis MG1363 by electrotransformation,and the recombinant T7g10L-Usp45-pgs A-?C-e GFP-p MG36e/MG1363 L.lactis(abbreviated as MG1363/?C L.lactis)was successfully constructed.Fluorescence microscopy was performed on the recombinant L.lactis,and the recombinant L.lactis emitted green fluorescence under the action of blue excitation light,which indicated that the ?C protein was successfully expressed on the cell surface of the recombinant L.lactis.The approximately 80 k Da ?C fusion protein was identified by Western Blot.The results showed that the NDRV ?C fusion protein was successfully expressed in LAB and the protein was anchored to the cell wall through the transmembrane anchoring domain.Part 2: Evaluation of the immune effect of the recombinant MG1363/?C L.lactis expressing ?C proteinTwo-day-old ducklings were orally immunized for 7 days with the MG1363/?C L.lactis,MG1363/p MG36 e L.lactis and MG1363 L.lactis respectively,and the blank group was used as a negative control.The levels of NDRV specific antibodies(including Ig G in sera and s Ig A in small intestinal fluid),and cytokines in sera were detected by ELISA and evaluated the immune effect.The results showed that the NDRV specific-Ig G in sera and total s Ig A in small intestinal fluid were significantly increased in the MG1363/?C L.lactis group.The levels of IL-2,IL-4,IL-10,and IFN-? were significantly increased compared with the other control groups(P < 0.05).On the eighth day,two ducklings injected with NDRV virulent strain were respectively assigned to each group to simulate natural infection and the mortality of the ducklings in each group was monitored.The result showed that the protection rate of the ducklings in the MG1363/?C L.lactis group was 76.67%.The LAB live vector vaccine constructed in this study provided a new way for the prevention and control of NDRV in the future.