| Reticuloendotheliosis(RE) is a tumorous disease of turkey, chicken, duck, partridge, pheasant, goose caused by reticuloendotheliosis virus(REV),which is characteristic of hyperplasia of lymphocytic netted cell. The lethal function of REV is not obvious to birds,but REV can cause the bursa to wither, its pathogenesis gene V-rel can cause the Blymphocyte to transform as the tumor cell, damage the birds organism immunity system, and cause organism resistivity to drop, thus the gene can cause other concurrent diseases and create a high elimination rate and a high mortality rate. So, it is urgent to establish a method to detect and control this disease.In this study,the env gene of REV was cloned and the env protein was induced to express by molecular biology method.Then REV was resplaced by the recombinant and purified GST-env protein as antigen to coat micro-plate of 96 wells, an indirect ELISA assay(ienv-ELISA)was developed and compaired with a REV-based ELISA diagnostic reagent kit(iREV-ELISA) to detect the accuracy rate. The result show that the accuracy rate was vary high and proved our country's poultry production a special, sensitive, safty, rapid and inexpensive domestic kit to detect REV antibody.In order to obtain recombinant GST-env protein, an expression plasmid pGEX-4T-3-env was constructed and transformed into E.coli BL21 strain,and IPTG was used to induce env protein. The cytorrhyctes was broken into pieces and solved by ultrasound wave and 8mol/L urea respectively.After renaturation with urea at 4℃,the expressed fusion protein GST-env was purified by GST protein purification kits.The content of purifed protein was 391.98μg/mL by detection,and western blot test proved that the purified protein had good antigenicity and reactogenicity.This research used the recombinant and purified GST-env protein as antigen to coat micro-plate of 96 wells, an indirect ELISA assay(ienv-ELISA)was developed to detect anti-REV sera. The optimum recation conditions for ienv-ELISA were investigated, and the result showed: optimal coating buffer was CB;optimal confining liquid was 1%BSA-PBS;optimal concentration of antigen was 8μg/mL;optimal dilution of sera was 1:300;optimal reaction time of sera was 1h;optimal dilution of HRP-labled goat anti chicken IgG was 1:5000;optimal reaction time of HRP-labled goat anti chicken IgG was 1h;optimal reaction time of substate was 25min. Repeatability tests REVealed that the coefficients of variation of positive sera within and between runs were less than 15%. Cross-reactivity assay showed that this assay was REV-specific.This assay was validated by comparison with a REV-based ELISA. The diagnostic sensitivity (DSN), specificity (DSP) and accuracy of the ienv-ELISA were 90.625%, 93.75% and 92.7% respectively.235 sera samples which came from some chicken farms in Shandong were detected by the ELISA kit. The result indicated that there were 77 positive serum samples,158 negtive serum samples,and the positive rate of REV antibody was 32.77%, and the accuracy rate was 90.2% compaired with the iREV-ELISA... |
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