Prokaryotic Expression Of P23 Major Surface Protein From Theileria.Sergenti

Theileriosis is a blood protozoal disease caused by Theileriidae, Theileria,Theileria sergenti.Recent years,Theileriosis result in economic losses seriously in animal husbandry with rising tendency,and paied the worldwide's attention.23-ku piroplasm surface proteins(p23)of T.sergenti was reported one of immunogenic protein and expressed on the surface of the merozoite membrane.A pair of specific oligonucleotide primers designed according to the gene encoding p23 surface protein of Theileria sergenti(D84447),and cloned 507 bp fragment from genomic DNA of T.sergenti. Homology of nucleotide sequence between the cloned p23 gene and the published sequence in GenBank was 99.4%,which proved that the purpose gene fragment was p23 surface protein of Theileria sergenti.Then p23 DNA was ligated into pGEX-4T-1 vector and was identified by PCR,digestion with endonuclease and sequencing,which proved to constructed a prokaryotic expression plasmid pGEX-4T-p23 successfuly,then transformed pGEX-4T-p23 into E.coli BL21 and inducd by -IPTG.,expressed approximately 46 ku fusion protein.Optimal expression conditions were determined by SDS-PAGE.The optial incubation time was 2.0h after inoculation,the optial induction time was 6.0h,the best temperature was 34℃,and the optimal concentration of IPTG.was 0.08 mmol/L.Under the optial expression conditions,the fusion protein account 31.7%of the total bacterium protein by Bandscan5.0.Western-blotting result showed that the protein can recognize positive serum of Theileria sergenti and has good reactionogenicity.