Immunogenicity Study Of Eimeria Tenella Calcium Binding Protein

Chicken coccidiosis is an obligate parasitic disease caused by chicken coccidia in the small intestine and cecal epithelial cells of chickens,causing huge economic losses to the poultry industry.Both drug control and vaccine prevention measures are unable to achieve the desired effect in the prevention and treatment of coccidiosis.Therefore,the search for a protein with better antigenicity to develop a cheap and efficient new vaccine has become the key to the prevention and treatment of coccidiosis.In this study,the ETH₀0009450 gene was screened by proteomics analysis,and the expression of this gene was up 1.569 times when Eimeria tenella invaded DF1 cells.This study selected this gene for research.First,a series of biological analysis of the EtCaBP gene was carried out.The analysis of the secondary and tertiary structure of the amino acid sequence of the gene showed that the expressed protein of the gene was calcium binding protein?CaBP?and had a characteristic EF-hand calcium binding domain.They can interact with calcium ions and play an important role in various life activities such as intracellular calcium signaling and calcium homeostasis.Therefore,it is speculated whether the gene is related to the invasion of host cells by coccidia,so as to study the immunogenicity of the protein and provide screening for candidate proteins for the study of novel vaccines against chicken coccidiosis.After analyzing the biological information of EtCaBP?Eimeria tenella calcium binding protein?,the gene fragment was obtained by PCR amplification,and then digested with NcoI and Hind III and cloned into prokaryotic expression vector pET32a-EtCaBP,which was transformed into E.coli expression.In the strain BL21?DE3?,the EtCaBP protein was induced and purified by IPTG,and the subunit vaccine was prepared.The mouse polyclonal antibody was prepared by immunizing the mouse.Construction of a yeast surface display system The EtCaBP protein was displayed on the surface of the yeast to identify polyclonal antibodies prepared.In this study,the immune effect of the EtCaBP subunit vaccine was evaluated by the chicken coccidial infection test to explore the immunogenicity of the EtCaBP protein.120chicks with small differences were randomly divided into group A?EtCaBP immuno-infected coccidia group?,B?EtCaBP immunized non-infected coccidia group?,C?non-immunized coccidial group?,group D?not immune to infection?Coccidial group),30 in each group.At 7days of age,the protein emulsified with complete Freund's adjuvant was administered to the A and B groups for multiple subcutaneous injections in the neck.The immunization volume was 100ug/only.The C and D groups were injected with the same amount of PBS;14 days old.At the same time,the second booster was immunized with incomplete Freund's adjuvant emulsified protein,and the immunization method was the same as 7-7 days old;at 21 days of age,10,000 fresh E.tenella sporulated oocysts were inoculated into groups A and C.The results showed that the EtCaBP protein has no transmembrane region but has a signal peptide sequence.The secondary structure prediction shows that it mainly consists of?-helix and contains five EF-hand calcium binding structures.It is predicted by tertiary structure and found to be CBP40(Physarum polycephalum specific Ca²⁺-binding protein LAV1-2)was structurally similar with a similarity of 66%.In this study,the EtCaBP gene was successfully cloned,and the prokaryotic expression vector was constructed to express soluble protein,and the purified protein was successfully used to prepare mouse polyclonal antibody.At 31 days of age,the data were analyzed and the results were analyzed by comparing the weight gain,relative weight gain rate,oocyst output,cecal disease value,anticoccidial index,lymphocyte transformation,cytokine fluorescence quantitative analysis and other indicators to EtCaBP.The immunoprotective effect of the subunit vaccine was evaluated.The relative weight gain rate,oocyst value,cecal lesion value and anticoccidial index of the EtCaBP immunization group were 88.9%,10,23.7,and 155.20,respectively.The results showed that the inhibition of EtCaBP protein to a certain extent can reduce the inhibition of E.tenella infection on chicken weight gain,reduce the oocyst discharge and reduce the degree of disease,anti-coccidial effect is effective.In summary,EtCaBP protein has a characteristic EF-hand calcium binding domain of calcium binding protein,which has certain immunogenicity and has certain immunoprotective effect on chicken infected coccidia.