| Chicken coccidiosis is an intestinal infection caused by intracellular protozoan parasites belonging to several different species of Eimeria. It is a severity disease of chicken, specially in modern intensive chicken farms. The characteristics of coccidiosis are fast propagation and difficulty to eliminate completely. It mainly infects chickling and cause high mortality, or lowers the production performance of chicken. Currently, this disease is mainly controlled through prophylactic coccidiostats and live vaccines. The extensive use of chemical drugs has resulted in the development of drug resistance of Eimeria spp, drug residue problem also limits the use of coccidiostats. Although live vaccines are effective to some extend for the prevention of the disease, the safty problem like virulence recovery and diffusion of the parasites limited the application of live coccidiosis vaccines. The genetically engineere vaccine is considered the most promising and safest vaccine to control the disease. Effectively protective antigens must be found out in the research and development of genetically engineered vaccine. Surface antigens of Eimeria play important roles in parasites pathogenicity and immunogenicity, some of which are potential candidate genes of vaccine or drug target. A great of research showed that most protective antigen genes are surface antigens of sporozoite and merozoites and organelles antigens.There are the most endogenous antigens at the stage of merozoites, which can induce host innune reactions. Result showed that the surface antigen of merozoite are glycosylphosphatidylinositol (GPI)-linked surface proteins, which have an N-terminal hydrophobic signal peptide, a C-terminal hydrophobic GPI signal-anchor peptide and an extracellular domain organized around six cysteine residues. But the roles in host adapted immunity of the complex repertoire of variant surface antigens of merozoite remains unclear. Therefore, genes of surface antigen17and18and unknown protein HP were cloned, analyzed and then expressied in E.coli, in this study establish foundations for succeeded studies of their immunogenicity and development of genetically engeneered vaccines in the future.Expression vector is an important tool for protein expression. A dual-function expression vector named pELI3.3was constructed basised on pCDNA3.1by meas of getting rid of BglⅡ cutting off SV40,Neomycin sequence, adding KOZAK sequence, ATG, MCS and.6His tags.This vector can be used for not only prokaryotic expression, but also eukyrotic expression in Expression library immunization (ELI) screening of protective antigens of E.tenella in succeeded study.Specific primers were designed according to the sequences of E.tenella Houghton strain and the genes of SAG17, SAG18and unknown protein HP of E.tenella Rongchang strain were amplified through RT-PCR from purified merozoites cloned and sequenced. Sequence analysis showed that both the nucleotide sequence and deduced amino acid sequence of SAG17, SAG18and HP gene to that of Houghton strain were higher than99%. This indicated that the three genes are highly conserved among different geographical strains.The cloned genes without signal peptide sequence were sub-cloned into the expression vector pELI3.3and transformed into E.coli strain BL21. All three genes were expressed through IPTG induction after optimization of expression conditions. SDS-PAGE showed that the band of expressed fusion protein of SAG17, SAG18and HP were at the poison of29.0kDa、29.0kDa、20.1kDa that matchs the theoretical molecular weight of them (27.76kDa、27.09kDa、19.3kDa respectively). The three recombinant proteins were mainly expressed in the form of inclusion body, and the expression level were up to28%、21%、27%of the total protein of host cells respectively. |
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