Expression Of S07-IL-2 And NA4-IL-2 Recombinant Yeast Protein And Evaluation Of Its Immune Effect

Coccidiosis is one of the most common and difficult to control important parasites on chickens,which brings serious harm and heavy losses to the poultry industry.At present,chicken coccidiosis is mainly controlled by chemical drugs,but its practical application is greatly limited due to drug tolerance and drug residue.In recent years,the genetic engineering vaccine and nucleic acid vaccine for the prevention and treatment of coccidiosis have attracted great attention from scholars at home and abroad.And Pasteur pichia expression system is a eukaryotic expression system has been widely used in recent years,has the genetic operation is simple,fast growth,high quantity of protein expression and fermentation advantages of low cost and express product is relatively easy to purification,at the same time,the system also have protein translation after the modification effect,such as glycosylation modification and function of protein phosphorylation,etc.In order to study the expression of recombinant yeast protein of e.enella in chicken and detect its immune protection,this experiment used the protective antigen gene SO7 of e.enella.NA4 gene and chicken il-2 gene were concatenated in eukaryotic expression vector pPICZ?A,and the recombinant plasmid pPICZ?A-SO7-IL2 ?pPICZ?A-NA4-IL-2was constructed.The constructed recombinant protein was immunized with whole bacteria fragmentation and induced expression protein,respectively,to evaluate the immune protection effect against tender ella emmellii.In this paper,the development and application value of yeast vaccine were reviewed,in order to provide theoretical basis for the research on the tender emeriococcidia vaccine.The main research contents are as follows:(1)Construction,expression and identification of the recombinant plasmid pPICZ?A-SO7-IL-2 and pPICZ?A-NA4-IL-2: the SO7 gene NA4 gene and the chicken IL-2 gene were amplified by PCR,and then the two genes so7-IL-2 and na4-IL-2 were connected to the pmd18-t cloning vector,respectively.Finally,so7-IL-2 and na4-IL-2 genes were connected to pPICZ?A vector.The recombinant plasmid was identified by PCR,double enzyme digestion and sequencing,and the results were confirmed.Methanol induced expression was finally verified and analyzed by Western blot.Meanwhile,pPICZ?A-SO7,pPICZ?A-NA4 and pPICZ?A-IL-2were constructed as controls.The results showed that the SO7,NA4 and IL-2gene lengths were 657 bp,252 bp and 468 bp,respectively.The recombinant plasmids pPICZ A-SO7-IL2 and pPICZ A-NA4-IL-2 were determined by PCR,double digestion and sequencing.Western blot results showed that pPICZ?A-SO7-IL-2 showed distinct bands of different concentrations at 42 KDa and pPICZ?A-NA4-IL-2 at 26 KDa with different induction time,and the band size was consistent with the expectation,which proved that the recombinant plasmid was successfully expressed in the yeast system.(2)The immune effect of the recombinant proteins pPICZ A-SO7-IL2 and pPICZ A-NA4-IL-2 against s.emmellii:The constructed recombinant proteins pPICZ A-SO7-IL2,pPICZ A-SO7,pPICZ A-NA4-IL-2,pPICZ A-NA4,pPICZ A-NA4 and pPICZ A-IL2 were immunized with intramusculus injection of chicken at 7,21,and 35 day immunization doses.Antibody levels and cytokine levels were determined by ELISA and T lymphocyte proliferation was determined by MTT assay.The results showed that at the levels of IL-2 and INF-?,the pPICZ A-SO7-IL2 and pPICZ A-NA4-IL-2 groups had A more significant difference than that of the control group(P < 0.01).At the level of T lymphocyte proliferation,pPICZ A-SO7-IL2,pPICZ A-NA4-IL-2 immunization group was significantly higher than pPICZ A-SO7,pPICZ A-NA4,pPICZ A-IL2 control group(P < 0.05).The recombinant protein was used to immunize chickens with a challenge test.The immune evaluation indexes included survival rate,relative weight gain rate,cecal lesion score,gram fecal sac number(OPG),lesion reduction rate and coccidiosis resistance index(ACI).The results showed that pPICZ A-SO7-IL2,pPICZ A-NA4-IL-2 immunization group than pPICZ A-SO7,pPICZ A-NA4,pPICZ A-NA4,pPICZ A-IL2 control group weight gain,cecal lesions reduced,the number of oocyst decreased.ACI reached 175.48,NA4 to be determined,showing good anti-coccidiasis effect.In this study,pPICZ?A-SO7-IL-2and pPICZ?A-NA4-IL-2 were successfully constructed,and their immune effects were confirmed by clinical trials,which laid the foundation for further development of anti-coccidiasis vaccine with practical value.