| Pocrine encephalomyocarditis is an acute infectious disease of swine caused by encephalomyocarditis virus(EMCV)and characterized by encephalitis, myocarditis or inflammation around cardiac muscles. In this study, first, a pair of specific primer to EMCV nonstructural protein 2C gene was designed and synthesized and 2C gene sequence of 1062 bp was obtained by RT-PCR. Moreover, recombinant expression plasmid pGEX-6P-2C was constructed and predicted recombinant nonstructural protein 2C of 62 ku was expressed in E.coli BL21 cells at the rate of about 33.9%. No visible degradation was found when recombinant protein 2C was kept in room temperature for 72 h, -20℃and -80℃for12 months, or when it underwent freeze- thawing for 9 times. And Western blotting showed that recombinant protein 2C could be recognized by positive pig or rabbit anti-serum to EMCV.Second, an indirect ELISA was developed to detect antibody against EMCV with purified recombinant protein 2C as coating antigen, reaction condition and working concentrations optimized, cut-off value confirmed, specificity, sensitivity and repetition of assay detected, and serum antibody against protein 2C dynamically analysed. Results indicated that the cutoff value of ELISA was 0.27; optimal reaction condition and working concentration went as follows: incubating the coating 100μL of recombinant antigen (1μg/mL) at 37℃for 1 h and at 4℃for overnight; blocking the uncoated part at 37℃for 1 h with PBST containing fetal bovine serum of 5%; diluting serum sample or the HRP conjugated to secodary antibody (goat anti-pig IgG-HRP) with blocking buffers at 1:20 or 1:400 and incubating it at 37℃for 1 h or 45 min; reacting with HRP protected from light at room temperature for 15 min after adding substrate. Moreover,recombinant 2C protein had no cross-reaction with positive sera of other 4 swine diseases (CSF, PCV2, PPV , PR and PRRS infection); EMCV could block the specific combination of recombinant antigen with positive serum of pocrine encephalomyocarditis. The concordance between this ELISA and neutralization test was 95.7% (22/23), and the relative sensitivity was 90.9% (10/11). The coefficient of variant (CV) in repetition test at the same time was under 10%, and that at different times under 15%. Antibody against EMCV was detected in sera of piglets on 4 d post infected (dpi). The antibody reached to the highest on 7 dpi, and then gradually declined but lower antibody to 2C could still be detected until 21dpi.Third, 1306 serum samples collected from different areas in Hebei Province were detected by the indirect ELISA. The results showed that an average positive rate of antibody to EMCV was 13.48%, in which the highest antibody positive rate occurred to postpartum sows (27.68%), the secondary to gilts and suckling pigs (16.67% and 2.86%) and the lowest to growing pigs (1.98%).In summary, the recombinant EMCV 2C protein was of high stability and reactinogenicity.Using it as antigen, indirect ELISA could be applied to early diagnosis of pocrine encephalomyocarditis and seroepidemiological survey. At swine farms of Hebei Province, EMCV infection had occured with a high infection rate in postpartum sow.
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