Screening And Expression Of Microneme Protein Gene 5 Of Eimeria Stiedai And Immunogenicity Of Recombinant Protein

Occidiosis is a kind of sporozoal diseases in rabbits caused by coccidian of Eimeriidae, which has posed a great threat to rabbit breeding industry in our country. In this paper, the following studies on investigation of coccidia species of rabbits in lanzhou, isolation of pure specie of E. stiedai by coccidia-free rabbit and single oocyst techniques, construction of cDNA library of sporozoites of E. stiedai, screening and expression of microneme protein 5 (Es MIC-5) to assay immunogenicity of recombinant protein MIC-5 were conducted. The results can be summarized as follows:Firstly, 14 coccidia species of Eimeria were observed in this survey, which were E. stiedai, E. media, E. magna, E. irrestidua, E. piriformis, E. exigua, E. intestinalis, E. coecicola, E. neoleporis, E. nagpurensis, E. perforans, E. leporis, E. flsvescens and E. matsubayashii. The dominant coccidian species in rabbits are E. perforans(21.2%), E. irrestidua(10.0%) and E. stiedai(9.0%), respectively. The infection rate of Eimeria coccidian was 100 percent for rabbits of 1-4 months of age.Secondly, the cDNA library of sporozoites of E. stiedai was successfully constructed, which consisted of 6.2x10⁵ independent clones with a recombination rate of 94.9%. The inserted fragments were 500～1500bp in length.Thirdly, two positive plaques which harboring 1095 bp cDNA fragment were screened by the Benton-Davis hybridization technique from the cDNA library. Sequence analysis revealed that the gene fragment shared 89.1% identities in nucleotide sequence with the same region of Et MIC-5 gene, harboring 903 bp encoding region, 174 bp 3' UTR and 17 bp poly(A) tail. 4 apple domains (A-domains) were found in its deduced amino acid sequence.Fourthly, Es MIC-5 was successfully expressed in E. coli transformated by prokaryotic expression vector pETMIC-5. The expressed recombinant protein MIC-5 was purified with Ni²⁺ ion column.Fifthly, rabbit IFN-γgene was amplified by RT-PCR. The recombinant IFN-γwas expressed in BHK transfected by eukaryotic expression vector pVAXIFN-γwith liposome.Sixthly, MIC-5 protein was successfully expressed in Pichia pastoris in the form of secretion protein and proved to be a good immunogenicity in immunized mice.Last, based on the immunization of twenty coccidia-free rabbits, the results showed that the recombinant protein Es MIC-5 could efficiently induce specific humoral and cellular immune responses against E. stiedai.