| Chicken coccidia is a protozoa parasitized in chicken intestinal epithelial cells.It not only affects the growth and development of chickens,but also causes serious economic losses for livestock and poultry industry.Among them,the most pathogenic is Eimeria tenella,which is parasitic in the chicken cecum.Because its schizonts proliferate in cecal epithelial cells,it leads to cecal swelling,intestinal mucosal damage,and epithelial thickening.The disease is reported to cost billions of dollars in economic losses worldwide each year.In the process of contemporary animal husbandry production,due to the abuse of various drugs,not only caused the harm of drug residues and drug-resistant insects,but also the elimination of a large number of probiotics,thus breaking the balance of normal flora in the intestinal tract of livestock and poultry.Lactobacillus occupies a dominant position because of its green,safe,probiotic effect and the role of presenting foreign antigens.It also has a very broad prospect to use Lactobacillus as a live carrier of a new anti-coccidia vaccine.Because coccidia has a very complex life history,and an antigenic protein is only expressed in some stages and has periodicity,it may be that the new anti-coccidia vaccine based on a single antigen can only provide partial immune protection.Therefore,it is urgent to find a rapid and effective solution to improve the immunogenicity of antigen.In order to improve the immunogenicity of the antigen,by studying the life history of chicken coccidia and the expression of its antigenic protein,this topic selects different antigenic proteins expressed in different stages of coccidia,analyzes their protein amino acid sequences,and selects the antigenic fragments with good immunogenicity to construct the fusion protein recombinant Lactobacillus vaccine,so as to improve the immunogenicity of the antigen and provide a choice for the development of a new anti-coccidia vaccine in the next step.The specific research contents are as follows:In this study,four effective antigenic proteins,EtMIC1,EtMIC3,EtProfilin and EtGam56,expressed in the invasion stages of sporozoite,merozoite and gametophyte,were selected from Eimeria tenella with high pathogenicity.According to the research report,EtMIC1,EtMIC3,EtProfilin and EtGam56 are Eimeria tenella proteins,which have certain protective power against chicken coccidiosis.Among them,EtMIC1,EtMIC3 and EtProfilin proteins are expressed in the sporozoite and merozoite stage of chicken coccidia,and EtGam56 protein is expressed in the gametophyte stage.In this study,the amino acid sequences of the respective proteins were analyzed by DNA Star and other analysis software.Combined with the published reports on the antigenicity of these four proteins,the antigen fragments with good immunogenicity were selected as the candidate antigenic genes of recombinant Lactobacillus live vector vaccine.In this study,specific primers were synthesized according to the gene sequences of four antigen proteins（EtMIC1,EtMIC3,EtProfilin and EtGam56）provided by Gen Bank.The selected gene sequences were amplified by PCR.The amplified gene sequences were linearly connected with pET28a vector by homologous recombinant enzyme.The bacterial solution PCR detection and sequencing identification were correct.The results showed that the recombinant prokaryotic expression plasmid pET28a-EtMIC1-EtMIC3-EtProfilin-EtGam56was successfully constructed.In this study,the MG（EtMIC1-EtMIC3-EtProfilin-EtGam56）tandem fragment was amplified by PCR with the constructed recombinant prokaryotic expression plasmid as the template,and the P32-murO-MIC8-FLAG-CPE plasmid skeleton was amplified by PCR with the PMFC（P32-murO-FLAG-CPE）plasmid constructed in the laboratory.The MG tandem fragment was connected with the PMFC plasmid skeleton by homologous recombinase to construct the PMFC-MG（P32-murO-MG-FLAG-CPE）recombinant plasmid,Then,the recombinant Lactobacillus live vector vaccine PMFC-MG was constructed by electrotransfer into Lactobacillus expression live vector,and the recombinant Lactobacillus live vector vaccines PMFC-EtMIC1,PMFC-EtMIC3 and PMFC-EtProfilin were constructed by the same method.The protein expression was identified by SDS-PAGE,Western blotting and IFA.The results showed that four recombinant Lactobacillus vaccines were successfully constructed.The surface expression rate of fusion protein PMFC-MG-NC8 was 73.26%,that of recombinant protein PMFC-EtMIC1-NC8 was 51.15%,that of recombinant protein PMFC-EtMIC3-NC8 was 46.66%,and that of recombinant protein PMFC-EtProfilin-NC8 was48.28%.The results showed that the four recombinant Lactobacillus vaccines could express the recombinant protein,and the expression rate of PMFC-MG-NC8 was the highest,which was significantly different from the recombinant protein（P<0.05）,indicating that the fusion protein had higher protein display efficiency.In order to study the immune protection effect of recombinant Lactobacillus vaccine with fusion protein,we purchased 1-day-old non immune Hailan Brown chick cocks for animal immune protection test.The experiment was divided into 7 groups with 20 rats in each group.Groups Ⅰ and Ⅱ were PBS control group.Groups Ⅲ,Ⅳ,Ⅴ,Ⅵ and Ⅶ were orally immunized with Lactobacillus NC8,recombinant Lactobacillus PMFC-EtMIC1-NC8,PMFC-EtMIC3-NC8,PMFC-EtProfilin-NC8 and PMFC-MG-NC8 respectively.From the first day,chickens in groups Ⅰ and Ⅱ were orally inoculated with 500μL PBS every other day,the other groups were inoculated with the same dose of recombinant Lactobacillus（2×10~9CFU/mL）until 14 days,all except group Ⅰ were orally inoculated with fresh E.tenella sporulated oocysts for 1.5×10~4.The immune protective effect was evaluated by measuring the average weight gain,cecal lesions,oocyst excretion,ACI and serum mucosal antibody.The animal test results showed that compared with the non immune group infected with coccidia（Group Ⅱ）,the body weight of the fusion protein immunized group increased significantly,and the cecal lesion score and the number of fecal oocysts per gram decreased significantly（P<0.05）,indicating that the fusion protein recombinant Lactobacillus can not only alleviate the growth retardation and cecal pathological damage caused by E.tenella,but also effectively inhibit the invasion,growth and reproduction of chicken coccidia;The ACI value of the fusion protein immunized group was the highest,up to 175,which was significantly different from that of the non immunized group（Group Ⅱ）（P<0.05）;In terms of lymphocyte proliferation and the ratio of CD4+/CD8+in peripheral blood lymphocytes,the ratio of CD4+/CD8+in the fusion protein immunized group was significantly higher than that in the non immunized group（Group Ⅱ）.The results showed that the fusion protein could effectively induce cellular immunity;In terms of serum specific antibody IgG and mucosal immune antibody SIgA,the titer levels of serum antibody IgG and mucosal antibody SIgA in the fusion protein immunized group were significantly higher than those in the non immunized group infected with coccidia（Group Ⅱ）,indicating that the fusion protein can cause effective humoral immunity and produce higher serum and mucosal antibodies,The results showed that the fusion protein recombinant Lactobacillus vaccine could provide effective anti-coccidia immune protection against Eimeria tenella infection in chickens.The above studies show that using probiotics such as Lactobacillus as live vectors to express coccidia antigen protein on its surface,so as to construct recombinant live vector vaccine,which can stimulate the body to produce high humoral and cellular immunity,so as to significantly improve the immune protection of the body,which is of great significance in the prevention and treatment of chicken coccidia infection.These studies will provide important theoretical reference for the research and development of new anti-coccidia vaccine in the future. |
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