Prokaryotic Expression Of The JL-2Gene Of The Eperythrozoon Suis And Establishment Of Indire CT Elisa Based On Recombinant Protein

Eperythrozoonosis is caused by Eperythrozoon suis which parasitize in the plasma, the surface of erythrocyte or invades porcine erythrocytes of the swine. It is an infectious disease which the main clinical symptoms are anemia, jaundice and febrile.At present, there is no effective methods to prevent and cure the Eperythrozoonosis. The available investigative gene of the Eperythrozoon suis which provide by the National Center for Biotechnology Information (NCBI) are rarely. Therefore, the experiment has been screening from the constructed Eperythrozoon suis genomic library. By agarose gel electrophoresis and sequencing analysis, obtain a gene fragment which total length is929bp and no homology with corresponding sequences in NCBI. Application ORF Finder (Open Reading Frame Finder) analysis revealed that the sequence contains a438bp open reading frame and encodes145amino acids. It is no homology with known Eperythrozoon suis sequences and tentatively named as "Eperythrozoon suis JL-2". There is not found homology protein by protein homology analysis. So conjecture that the protein is likely to be a Eperythrozoon suis-specific protein.According to the largest ORF of the Eperythrozoon suis JL-2gene, a pair of specific primers were designed whit double endonuclease digestion sites and amplified by PCR. Then cloned it to the prokaryotic expression vector pGEX-4T-1and transformed into E.coli BL-21competent cells. It was highly expressed proteins encoded by the new gene after induced by IPTG and analysis by SDS-PAGE and western blotting. The recombinant protein molecular weight is40.5kDa. It can be identified by positive serum of the Eperythrozoon suis and has better reactogenicity.The experiment established the indirect ELISA for detecting the Eperythrozoon suis which utilized the purified of the recombinant protein as the antigen. By the reaction conditions of the ELISA screened, the best package concentration of the antigen was determined to be20.5mg/mL, overnight at4℃. The testing serum dilution was1:80and reacted at37℃for1h. HRP-rabbit anti-pig IgG dilution was1:2000and reacted at37℃for1h. The optimal reactive time of the substrate was15min at37℃. By the specificity, reproducibility and clinical serum samples test further showed that the establishment of the indirect ELISA has good specific, reproducible and high accuracy.The results showed that the study provides a new gene fragment for the Eperythrozoon suis further research and provides a basis material for the gene function studies, and also provides an simple economic, fast and effective way for the serological diagnosis of the Eperythrozoonosis.