Construction Of Recombinant Lactococcus Lactis Expressing VP1 Protein Of Duck Hepatitis Virus Type 3 And Immunity Test On Mice

Duck viral hepatitis(DVH)is an acute,highly contagious and deadly infectious disease in 3-week-old ducklings,which is caused by duck hepatitis virus(DHV).The clinical symptoms of the diseased ducklings are opisthotonos and liver lesions.DHVs can be classified into duck hepatitis A virus(DHAV)of the Picornaviridae family Avihepatovirus genus and duck astrovirus(DAstV)of the Astroviridae family Avastrovirus genus.DHAV is divided into three serotypes: type 1,type 2 and type 3.The infection of DHAV type 3(DHAV-3)has been prevalent in duck flocks in China,which caused great economic losses to the duck breeding husbandry.Thus,it is of great significance to pay more attention to DHAV-3.At present,people mainly use attenuated vaccine and inactivated vaccine to prevent the infection of DHAV-3 in ducklings.However,ducklings cannot be protected quickly and efficiently after using commercial vaccine.Oral vaccine of Lactobacillus living carrier has been widely used because of various advantages such as oral using,safety and non-toxicity.In this study,VP1 gene of DHAV-3,which expresses the major protective antigen protein of DHAV-3,was transmitted into a food grade lactic acid expression system.The recombinant expression system can express VP1 protein and be used as oral immune agents.This article is mainly divided into the following two parts:Part I: The construction of Recombinant Lactococcus lactis(L.lactis)system which can express VP1 Protein of DHAV-3L.lactis is one of the commonest probiotics in the intestinal tract of animals and humans.It is considered as a microbe of safety-level.The cellular immunity and humoral immunity can be activated by L.lactis.In this experiment,we choose L.lactis as expression system,which used food-grade L.lactis as a live vector to transmit VP1 protein and activate mucosal immunity.Firstly,the VP1 gene of DHAV-3 was amplified by PCR method.Secondly,VP1 gene was ligated with pNZ8149 vector(expression vector of the lactic acid bacteria)by fusion PCR technology and then constructed into the pEASY-T5 Zero vector(E.coli cloning vector).Next,the pNZ8149-VP1 was digested by single endonuclease and synthesized by T4 DNA ligase to transformed into a circular plasmid.Finally,the circular plasmid was transmitted intoL.lactis NZ3900.The recombinant L.lactis was induced by nisin to express protein and the expression product was demonstrated by Western blot.The result of Western blot showed no specific bands existed in the result of culture medium supernatant and ultrasonic disruption of the cells.However,a specific band of 29 kDa existed in cell sediment,indicating that the VP1 gene successfully expressed in recombinant L.lactis.Part II: Oral immunization of the recombinant L.lactis which can express VP1 proteinBALB/c mice were immunized by L.lactis that containing a recombinant plasmid pNZ8149-VP1 by oral method.The mice were immunized 3 days continuously every week and it were immunized 3 weeks in total.After immunized for the first time,three mice were selected randomly in each group to sample blood and intestinal mucosa in 10,20,30,37,44 and 51 days to analyze the effect of immunization in mice,we tested specific antibody of DHAV-3,conversion rate of lymphocyte and cytokine.The result showed that specific IgG and sIgA of intestinal mucosal can be induced in mice by immunized L.lactis that containing recombinant plasmid of pNZ8149-VP1.The lymphopoiesis was also increased.The number of IL-2?IL-4?IL-10 and IFN-? were significantly increased in experiment group in contrast with PBS group and blank control group.And the best effect of immunization happened at30 d.Studies indicated that the microecologics of recombinant L.lactis which can express DHAV-3 VP1 protein have broad prospects for the reason of great effect in humoral immuneresponse and cellullar immunologic-response.In the present study,the immunogenicity of the recombinant L.lactis expression system was evaluated by test index of humoral immunity and cellular immunity,which lays the foundation for the development of oral microecologics of L.lactis to prevent DHAV-3.