Construction Of A Live Vector BCG Vaccine Against Eimeria Tender Hebei Strain And Evaluation Of Its Immune Effect

Coccidiosis is a major parasitic disease that seriously harms the chicken industry.The disease is widely distributed,among which Eimeria tenioris is the most susceptible to infecting chicks,the infection rate is 60%～70%,and the mortality rate is up to 80%.After the recovery of the disease,the growth of chicks is hindered for a long time,and the performance of egg production is decreased,which brings great economic losses to the chicken industry.The most commonly used methods for preventing coccidiosis are pharmacotherapy and live vaccines,but both come with risks and limitations.Recombinant BCG vaccine is a type of live vector vaccine.It is a form of vaccine in which the antigen gene is attached to the expression vector of BCG vaccine and the plasmid is transferred into BCG vaccine,so that the live BCG bacteria can express the antigen protein in the host body.The purpose of this study is to construct recombinant BCG vaccine RBCGPMV261-IL-2-ETMIC-2 and verify its immune protection effect through animal immune protection attack insect test,which is divided into two stages.1.Construction of recombinant BCG vaccine PMV261-ETMIC-2-IL-2 and PMV261-ETMIC-2In this study,the total RNA of e.tenella hebei strain oocyst and chicken spleen was firstly extracted and reversely transcribed into c DNA.Using c DNA as template,THE ETMIC-2 gene and IL-2 gene were amplified by RT-PCR,and the recombinant plasmids PMD18-T-ETMIC-2 and PMD18-T-IL-2 were constructed.The recombinant plasmids pm V261-ETMIC-2 and PMV261-IL-2 were constructed by double enzyme digestion of the two recombinant plasmids,and then the two gene fragments were linked to the vector pMV261,respectively.The recombinant plasmid PMV261-ETMIC-2 was constructed by cutting the IL-2 gene from the recombinant plasmid p MV261-IL-2 and linking it to p MV261-ETMIC-2.BCG competent cells were prepared,and the conjugate products PMV261-ETMIC-2 plasmid and PMV261-ETMIC-2-IL-2 plasmid were respectively added into BCG competent cells.Recombinant BCG vaccine RBCGPMV261-IL-2-ETMIC-2 and RBCGPMV261-ETMIC-2 were constructed by electrical transformation.The recombinant BCG vaccine was successfully constructed and immunogenicity of the prepared ETMIC-2 antibody was verified by acid-fast staining and Western Blot.2.Study on the immune effect of recombinant BCG vaccine PMV261-ETMIC-2-IL-2 and PMV261-ETMIC-2 against Eimeria tender in chickenChickens were immunized with two kinds of recombinant BCG vaccine by nasal drip and oral administration,and another control group,red control group(attack insect control group)and white control group(blank control group)were set up to observe the immune protection effect.The results showed that the recombinant BCG vaccine et MIC-2 had immune protection against the attack of coccidiasis oocyst,and could produce corresponding cellular and humoral immunity.The anti-Coccidial index(ACI)of the red control group was only 79.28,suggesting that the virulence of the infected Coccidial oocysts was relatively strong,and the ACI of r BCGPMV261-ETMIC-2 nasal drip group and oral oral group were 164.63 and161.01,respectively.ACI of r BCBPMV261-IL-2-ETMIC-2 nasal drop group and oral oral group were 176.32 and 183.81,respectively.The anti-coccidiasis effect of all the four groups reached a good or above level,and the ACI of RBCGPMV261-IL-2-ETMIC-2 nasal drop group was 183.81,indicating excellent anti-coccidiasis effect.These results indicate that r BCGPMV261-MIC-2 and RBCGPMV261-MIC-2-IL-2 have good protective effects on e.tenella oocyst infection.In addition,the immune protection effect of RBCGPMV261-MIC-2-IL-2on E.temella oocyst infection was stronger than that of r BCGPMV261-MIC-2 alone,indicating that the combination of IL-2 could significantly improve the anti-coccidiosis immunity level of the vaccine,and the ACI of the nasal drip group was higher than that of the oral group.The results showed that the immune effect of nasal drops was better than that of oral administration.In this study,BCG live vector vaccines RBCGPMV261-IL-2-ETMIC-2 and RBCGPMV261-ETMIC-2 were successfully constructed,and the good immunogenicity was verified by animal immune protection attack insect test,which can effectively induce the immune response of the body.To a certain extent,the secretion of immunoglobulin and cytokines in the body is enhanced,and the T lymphocyte conversion rate is enhanced,so that the body has the ability to resist coccidioidal infection and injury,and the coccidioidal index has reached a good or above level.