Expression And Immune Effect Of ALV-J Gp85 Recombinant Lactobacillus Plantarum In Chicken

Avian Leukosis Virus Subgroup J(ALV-J)is a retrovirus subgroup that was first reported at the beginning of the 1990 s,when it was isolated from meat-type chickens.It has been the leading cause of marrow cell hyperplasia and renal tumor in white feather broilers.This disease has been widely reported in many countries and regions around the world,subjecting poultry industry to immeasurable risks,potential damages and serious economic losses.At present,foreign countries mainly rely on purifying the breeders to prevent and control ALV-J.However,it is impossible to completely eradicate ALV-J infection by means of sole purification,as the breeding environment has been contaminated mostly.To date,there are no commercial vaccines available for the prevention of ALV-J.Recently,numerous researchers have been dedicated to developing attenuated or inactivated vaccines against ALV-J.Nevertheless,it remains difficult to develop the ideal attenuated vaccine due to the complex antigen structure of the virus.Compared to attenuated vaccine,inactivated vaccine undermines its ability to induce the production of antibodies.Therefore,the development of new and effective ALV-J vaccine is imperative.As most of bacteria and viruses attack body through mucosal invasion,it is effective to take advantage of a live vector that presents antigen to mucosal tissue so as to induce the mucosal immune response to resist invasion from a large amount of bacteria and viruses.In recent years,lactobacillus plantarum as an oral vaccine has become a hot spot.It is used as a live vector to deliver exogenous genes.Moreover,its beneficial function can be effectively combined with the immunogenicity of exogenous antigen.In addition,lactic acid bacteria is the normal physiological bacteria,thus this new vaccine is a relatively safe,convenient and cheap approach.In this study,we focused on the protective antibodies induced by the prokaryotic expression of avian leukemia virus subgroup J gp85 envelope protein with lactobacillus plantarum as the host strain,and investigated the process of anti-ALV-J infection in chicks,aiming to develop a safe and effective vaccine against ALV-J.To start with,by performing Polymerase Chain Reaction(PCR),we amplified the gp85 envelope gene and the pgs Atransmembrane protein gene from avian leukemia virus subgroup J env gene and the genomes of pgsA,pgsB and pgsC of Bacillus subtilis respectively.Then the gp85 and pgsA genes were cloned into the expressing vector pMG36 e with Emr labellings,which can shuttle and express in E.coli and L.casei.After that the recombinant vector pMG36e-pgsA-gp85 was transformed into the competent cells Lb.plantarum for expressing the target proteins we needed.The recombinant protein was examined by SDS-PAGE and Western-blot,and the expression of the protein on the surface of recombinant Lactobacillus plantarum was detected by flow cytometry.Next,SPF Hy-Line Brown layer chicks were orally immunized with the recombinant strain Lb.plantarum harboring pMG36e-pgsA-gp85 plasmids.After immunization,the chickens were weighed every week and the specific anti-ALV IgG in the serum of chicks were detected by indirect ELISA every week after the first immunization.Besides,the levels of IgA in the secretory solution were determined by ELISA.Finally,in seven weeks after immunization with the recombinant Lb.plantarum,all the chicks were challenged with the standard-type ALV-J(NX0101).The results showed that the levels of IgG and IgA rise significantly after immunization with the recombinant Lb.plantarum(pMG36e-pgsA-gp85/ Lb.plantarum)in chicks.The average viremia rate of the control group was 60.9%,while that of the oral recombinant Lb.plantarum group was 14.6%.Thus,the recombinant Lb.plantarum vaccine we constructed has a certain resistance to ALV-J.To the best of our knowledge,this is the first work that uses Lb.plantarum as the host strain to express the envelope protein gp85 gene of avian leukemia virus.Our study shows that the gp85 protein was stably expressed on the surface of bacteria by pgsA anchoring protein gene.The recombinant Lactobacillus plantarum we constructed showed considerably immunoprotective effect on ALV-J.In summary,our work not only provides the reliable experimental data and scientific basis for the development of the recombinant Lb.plantarum oral vaccine against subgroup J avian leukemia,but also could shed light on novel ideas and theoretical reference for the prevention and treatment of other capsular viral diseases.