Construction Of CDNA Library And Screening Of Antigen Gene Of Eimeria Acervulina Sporulated Oocysts

Avian coccidiosis is the major parasitic disease of poultry infected by Eimeria spp. It inflicts severe economic losses on the poultry industry. At present, conventional disease control strategies have relied on prophylactic medication and immunization with live vaccines. Due to the emergence of drug resistant parasite and the danger of live vaccines etc, Novel approaches are urgently needed to study to control coccidiosis. At present, identification important parasite antigen genes are crucial for the design of novel control approaches.At first, the traditional biological method was used to identify Eimeria acervulina species by some classical indexes, including prepatent period, location of coccidia in the intestine, the shortest sporulation time, size and shape of oocysts and sporocysts and so on. In order to screen antigen gene,we constructed the expression cDNA library of E. acervulina sporulated oocysts with SMARTTM cDNA library construction kit. All results showed that the titer of primary cDNA library was 4.6×10⁶ pfu/mL, the titer of amplified cDNA library was 4.4×10¹⁰ pfu/mL, the recombination rate was 98â„…, the average insert fragments was 1 000 bp. The antiserum of the rabbit immuned with the E. acervulina sporulated oocysts were used as a probe to screen cDNA expression library. Six positive clones were gained, PCR amplification revealed the length of no. 1³ positive clones was 1 000 bp, the length of no. 4⁶ positive clones was 1 500 bp.A novel gene including a complete open reading frame was obtained with RACE technique (GeneBank accession number: EU590120), which encodes a protein with 163 amino acids. With bioinfomatics analysis software, property and function of protein coded by the novel gene were predicted, including physical and chemical property, trans-membrane structure, antigen site, conserved domain, function site and homologous protein search, the results indicated that the gene encoding product contains one protein kinase c phosphorylation sites, one N-glycoylation sites, two casein kinaseâ…¡phosphorylation sites, fore N-myristoylation sites.The macrophage migration inhibitory factor (MIF) was obtained from the expression cDNA library of E. acervulina sporulated oocysts by PCR. The PCR product was purified and ligated to prokaryotic expression vector pET28a (+), the recombinant plasmids pET28a-MIF was constructed and transformed into BL21 (DE3) for expression. The fusion protein was purified successfully with Ni resin. Western-blot revealed that proteins were native antigens.The expression cDNA library of E. acervulina sporulated oocysts was constructed, a novel gene was screen with antiserum from the cDNA library. The MIF gene was obtained by PCR and expressed successfully. These results provided the foundation for cloning useful genes of genetically engineering vaccines and further studying new drugs to control coccidiosis.