| Pseudorabies virus(PRV), a member of the alp Haherpesviridae cause Aujezskey’s disease(AD), can produce fatal encep Halitis in newborn pigs, respiratory disorders in fattening pigs and reproductive failure in sows, resulting in swine devastating disease and economic losses worldwide. Although g E-null vaccination and g E-ELISA antibody test kit were effective in reducing the circulation of wild PRV and preventing the illness, it generally does not prevent infection for the reason of a broad range of vertebrates especial the wild pig and a long incubation period which can infect other health pig.In the wordwide, widespread using g E vaccine strains makes it possible to serologically distinguish g E-null vaccine strain-infected animals from virulent field strain-infected animals for the detection of antibodies to g E. A commercialization of PRV-g E ELISA kit is often used for DIVA diagnosis. But ELISA methods need specific equipment and a certain technical training for the staff in the lab not suitable for veterinarian to make fast diagnosis. Therefore, this study develop a fast and simple gold-immunochromatographic assay for the detection of g E antibodies against PRV.In this study,The DNA fragments encoding the PRV g E epitope(945bp)was amplified by polymerase chain reaction(PCR) from PRV genome. The purified PCR product was digested with Eco RI and Xho I, and cloned into the PET32 a vector. The recombinant plasmids of p ET-32a-g E, was ransformed into Escherichia coli(E. coli) BL21(DE3) competent cells. The target protein purified from supernatant by using Ni-NTA His·Bind Resin was analyzed by western bolt for the presence of g E. Purified PRV-g E and pig-Ig G were blotted on a nitrocellulose membrane for the test(T) and control lines(C), respectively. Stap Hylococcal Protein A(SPA) labeled with colloidal gold, was dispensed on a conjugate pad as the detector. Based on this, we developed the Gold-immunochromatographic Assay for the Detection of Antibodies against g E of Pseudorabies Virus. Criterion for judgement: if the tested serum contains the Ig G, antibodies against the PRV-g E, the Ig G will interact with the colloidal gold-SPA to form an complex(gold-SPA-swine Ig G). The complex will react with the immobilized PRV-g E on the T line and Pig-Ig G at the C line of the ICS to form two visible red band. If there is no Ig G antibody against PRV-g E in the sample serum, only the C line will be visible. We study the best reaction condition for the immunochromatographic Assay, finally determined that pig Ig G(2mg/ml) and PRV-g E(2mg/ml) were dispensed onto the C line and T line, the optimal conditions for conjugation between gold and SPA( 0.1 mg/ml) was p H 8.0, and serum samples diluted to 50 times was best for the ICS test.In this paper, we study specifically, sensitivity, repeatability and stability as well as the conformity of the ICS proved that the strip was capable of specifically detecting PRV-g E antibody and detected negative for other virus serums such as PRRSV, PCV, PPLO, PEDV and TGEV. The strip sensitivity can reach 250 times dilution degrees. Its stability and reproducibility were quite excellent after storage at 4°C within 4 months. Compared with IDEXX Pseudorabies Virus g E Antibody Test Kit(IDEXX PRV g E Ab Test), The strip sensitivity was slightly high and using the the IDEXX PRV g E Ab Test as a reference, the relative specificity and sensitivity of the ICS were determined to be 81.6% and 90.7%, respectively. Furthermore, there was an excellent agreement between the results obtained by the commercial product and the ICS(kappa = 0.7289). In this study, a total of 28 pigs were randomly divided into 4 groups: nonvaccinated and challenged(NVC) group(01), nonvaccinated and no-challenged(NVNC) group(02), vaccinated and challenged(VC) group(03), vaccinated and no-challenged(VNC) group(04). We monitored this experiment for 45 days proved that all serums detected by ICS and IDEXX test were emerged positive results at 9, 13, 9 and 13 dpc, which show good consistency between this two method.In conclusion, we develop a new gold-immunochromatographic assay for the detection of g E antibodies against PRV. |
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