| Coccidiosis is a class protozoa disease,which is caused by one or more Eimeria spp infect with intestinal cells.The Eimeria spp life history very complicated,it not only raises the nutrients in the intestine but also causes varying degrees of damage to the gut cells.At present,the prevention and control of coccidiosis is still dominated by drugs,however,prolonged use of drugs makes resistant strains appear constantly,therefore,coccidiosis problem becomes more and more intense.Over the years,researchers have tried to solve this problem by other ways,these include optimization of the use of anti-coccidiosis drug,screening of new anticoccidial drug and preparation of new anti-coccidiosis vaccine.Although these methods have long been reported,however,the culture of coccidiosis in vitro is not completely established and strains cross protection lower leading to more difficult research of coccidiosis drug screening and anti-coccidiosis vaccine.Therefore,the study was based on looking for the ideal protective antigen,cloned E.tenella surface antigen family genes EtSAG4,EtSAG16 and EtSAG22 which that play an important role in invasion and growth.Then,the three genes were separately or jointly inserted into the prokaryotic expression vector pET28 a and the eukaryotic expression vector pEGFP-N1.On this basis,animal experiments were conducted in order to evaluate the protective effects.The research mainly includes the following aspects:(1)Isolation and genetic polymorphism analysis of E.tenella wild isolatesInoculated the eight E.tenella wild isolates to chickens after oocysts sporulated,we collected oocysts from the cecum,utilized morphology and ITS1 identification method obtained the E.tenella wild strain from twenty one chicken farms.ITS1 sequence analysis showed that different regions E.tenella strains were highly conserved,and sequence rates range from 92.7% to 100%.The phylogenetic analysis of ITS1 sequence of four geographical regions and other countries showed that wild strains were also conserved with other countries.RAPD analysis of nine wild isolates in urban distribution showed that the genetic characteristics of the isolated isolates were local and cross-border cross-correlation.(2)Cloning and analysis of E.tenalla surface antigen genes EtSAG4,EtSAG16 and EtSAG22The full-length sequence of three genes was successfully amplified from cDNA of E.tenalla Suizhou 1 strain,and sequence alignment showed that three strains had 100% sequence homology with Houghton E.tenalla strain.Bioinformatics analysis revealed that amino acid sequences of three antigens contained a signal peptide at N-terminus and a GPI-anchored domain at C-terminus,and have less hydrophilic.(3)Prokaryotic expression of EtSAG4,EtSAG16,EtSAG22 and EtSAG41622The three genes partial sequences without signal peptide and GPI GPI-anchored domain were amplified from the full length gene,and make single gene or three genes were inserted into the prokaryotic expression vector pET28 a.At 28℃ for 10 h,recombinant protein EtSAG4,EtSAG16,EtSAG41622 were expressed,at 37℃ for 5 h,expressed recombinant protein EtSAG22.Western blot results showed that the four recombinant proteins could reacted with His-tag antibody and positive sera infected with E.tenalla for 15-day-old chickens,the results demonstrated expression of these four recombinant protein.(4)Eukaryotic expression of EtSAG4,EtSAG16,EtSAG22 and EtSAG41622The partial sequences of the three genes without signal peptide and GPI anchor domain were separately or co-constructed into the eukaryotic expression vector pEGFP-N1.Four plasmids were transfected into 293 T cells by lipofection method,fluorescence microscopy showed that the cells transfected with recombinant plasmids all could produced green fluorescence,however,the cells transfected with pEGFP-N1 plasmid also produced green fluorescence,which could not prove the successful transfection of the recombinant plasmids.The results of RT-PCR showed that the target gene of the four inserted fragments was amplified by using the cDNA of the transfected recombinant plasmid as a template,however,any fragment was not amplified by transfecting pEGFP-N1 plasmid as template and negative control.Western blot results showed that the recombinant plasmids transfected cell proteins could react with GFP tag antibody,however,negative control failed to react with GFP tag antibody.The recombinant plasmids was successfully expressed in 293 T cells by RT-PCR and Western blot.(5)Evaluation of immune protection effects of EtSAG4,EtSAG16,EtSAG22 and EtSAG41622In this study,the protective effect of E.tenella infection on experimental broilers was evaluated,immune recombinant protein animal experiment set 10 groups,immune recombinant plasmid animal experiment set 11 groups.Except for the control group,the experimental group animals were immunized with recombinant prokaryotic expression protein or recombinant eukaryotic expression plasmids in different doses(50 μg or 100 μg)at 14 days,2 immunizations were administered at 21 days of age,one week after the 2st immuned,each of the experimental animals was orally infected with sporulated E.tenella oocysts 5 × 104(except for non-infected and non-immunized group).Experiments were performed to detect cytokine levels,IgY concentrations and statistical data of coccidiosis index,so as to comprehensively evaluate the protective effects.Immuned recombinant protein animal experiments showed that: There was a significant difference in concentration of IFN-γ between the immuned recombinant protein groups and the infected non-immunized group.The groups immuned recombinant protein EtSAG22(100 μg),EtSAG16(100 μg)and EtSAG41622(100 μg)IL-17 concentrations were significantly different with the infected non-immunized group.The IgY concentrations between the immunized EtSAG16(100 μg)and EtSAG41622(100 μg)groups and the infected non-immunized group were statistically different.These results indicate that different doses of recombinant protein immunization could stimulated the body’s ability to produce IFN-γ levels.The ACI results showed that,the ACI of the three recombinant protein experimental groups reached more than 160,were 163.77 for the immunoproteins EtSAG4(100 μg),166.78 for the EtSAG16(100 μg)and 165.63 for the EtSAG41622(100 μg)group,respectively,the experimental groups that were immunized with the three doses of protein showed moderate against E.tenalla infection.Immuned recombinant plasmid animal experiments showed that: IFN-γ and IL-17 concentrations in the immunized recombinant plasmids and infected non-immunized group were significantly different.There was statistically significant difference in the concentration of IgY in seven dose groups of immunized recombinant plasmids and infected non-immunized group.The above results showed that different doses of recombinant plasmids could stimulated the body to produce IFN-γ and IL-17 levels.The ACI results showed that,the ACI of the six recombinant plasmids in experiment group reached more than 160,of which 173.11 was the highest in group of the immunized recombinant plasmid EtSAG41622(100 μg),EtSAG41622(100 μg)group 168.91 second,168.28 of the groupEtSAG22(100 μg)third,this result showed that the experimental groups immunized with these six doses of recombinant plasmids have the protective effect against E.tenalla infection. |
