| Porcine reproductive and respiratory syndrome virus (PRRSV) can cause reproductive failure in pregnant sows and respiratory distress in piglets. PRRSV is a small-enveloped virus containing a positive-sense (+), single-stranded RNA genome of approximately15.4kilobases in length, which codes13non-structural proteins (NSPs):Nspla, Nsp1β and Nsp2to Nsp12and6structural proteins:GP2, GP3, GP4, GP5, M and N. After infection of PRRSV in pigs, the antibody to Nsp7could be detected as early as14days post infection (dpi), and the responses lasted more than202dpi, which being equivalent to the immune response to N proteins of PRRSV. In the study, Nsp7of PRRSV SY0608isolate was expressed in E.coli and the purified recombinant protein was used as antigen in ELISA. And an indirect ELISA kit for detecting antibody to PRRSV was developed and used for epidemiological survey of PRRSV and evaluation of vaccine immune effect. The main contents were as following:1. Construction and stability of expression vector of Nsp7gene of PRRSVPRRSV SY0608isolate Nsp7gene was amplified by PCR and cloned into a prokaryotic expression vector pET-28a. A recombinant plasmid named pET-28a-Nsp7was constructed and identified with restriction enzyme digestion and sequencing. The recombinant fusion protein Nsp7was highly expressed in E.coli BL21with molecular weight of35KD. The stability of the expression vector was studied. Ten positive colonies were selected randomly and the Nsp7gene was confirmed by PCR. After the recombinant bacteria were passaged by thirty times, the recombinant protein Nsp7of the5th,10th,20th and30th generation were still expressed efficiently and confirmed by SDS-PAGE and Western blot. It indicated that the expression vector had a sound stability. 2. Preparation and detection of the quality of recombinant protein Nsp7In order to provide high quality coated antigen for the subsequent indirect ELISA, the recombinant protein Nsp7was largely expressed in E. coli BL21supernatant under the conditions of optimizing the expression conditions. Then expression products were purified using Ni-NTA affinity chromatography and the quality was detected by SDS-PAGE, Western blot and ELISA. The optimal initial induction concentration of the bacteria was0.8(OD6oonm), the optimal final concentration of inducer IPTG was1.0mM and the induction time was6h at37℃. The results of SDS-PAGE and Western blot showed that three batches of the purified recombinant Nsp7had clear target stripes without others ones and good antigenicity against anti-Nsp7monoclonal antibodies.The average concentration of three batches of the antigens measured by the spectrophotometer were0.354mg/mL,0.327mg/mL and0.299mg/mL, respectively. The coating concentration for ELISA of three batches antigens was0.40μg/mL. It indicated that the recombinant Nsp7could be used as the antigen in ELISA for detection of the antibodies to PRRSV.3. Preparation and quality detection of positive and negative control seraSwine PRRSV positive control sera were collected from21-35-day-old piglets free of PRRSV after infection experimentally with PRRSV strain SY0608-F40at a dose of1060TCID50by intramuscular injection route and S/P ratios detected by IDEXX ELISA Kit were more than0.4. PRRSV negative control sera were collected from28-day-old piglets, which were shown to be free of PRRSV, PCV2, PRV and CSFV antibodies. Positive control sera and negative control sera were satisfactory by the detection of character, sterility and the antibodies against common porcine viruses, which supplied good control sera for the subsequent ELISA method.4. Establishment of Nsp7indirect ELISA for detection of antibody against PRRSVAn indirect ELISA method was established with the purified recombinant protein Nsp7as the coated antigen through optimization in reaction conditions. The optimal antigen concentration and serum samples dilution were set at0.4ug/mL and1:100. The coated time was2h at37℃. The sealing buffer was1%BSA-PBST, and the sealing time was3h at37℃. The serum samples were incubated for1h at37℃. The dilution of the conjugate was defined as1:20,000and the reaction time was30min at37℃. The TMB substrate was added and incubated at37℃for10min before terminated with2M H2SO4. The cut-off value of ELISA was determined by detecting thirty PRRSV negative sera. When the serum samples with S/P ratios more than or equal to0.4were considered as positive, those with S/P ratios less than0.3were considered as negative, and the others were suspicious. The diagnostic sensitivity, specificity and accuracy of the Nsp7ELISA were90.0%,95.0%and92.0%, compared with IDEXX ELISA kit on50serum samples. Cross-reactivity assay showed that Nsp7ELISA was PRRSV-specific. It indicated that the Nsp7ELISA is not only sensitive, specific, and time-saving but also is suitable for large scale epidemiological surveys of PRRSV infection.5. Development and validation of indirect ELISA kit based on Nsp7for detection of antibody against PRRSVUsing three batches of the purified recombinant protein Nsp7and setting the negative and positive control sera by detecting30negative sera, indirect ELISA kits for PRRSV antibody detection were developed. Three batches of Nsp7ELISA kits were validated by sensitivity and specificity, repeatability and storage tests. The sensitivity of three batches of Nsp7ELISA kits were93.3%,93.3%and90.0%, compared with IDEXX ELISA kit on30positive serum samples, and the titer of10positive reference sera could be detected up to1:640. The specificity of three batches of Nsp7ELISA kits were all100%, compared with IDEXX ELISA kit on30negative serum samples, and there were no cross-reaction with the positive reference serum antibodies to PCV2, CSFV, PRV, EMCV, HPS and FMD.The intra-assay variation coefficient ranged between3.39%and8.27%, whereas inter-assay variation coefficient ranged between0.47%and7.30%, which showed a nice repeatability.Three batches of Nsp7ELISA kits could be stable at4℃for at least eight months. It indicated that an inexpensive, rapid, sensitive and specific diagnostic ELISA kit was deveploed, and it provided an available tool for antibody detection and serological survey of PRRSV.6. Application of an indirect ELISA kit for PRRSV-specific antibody detectionThe diagnostic sensitivity, specificity and accuracy of the PRRSV Nsp7ELISA kit were91.4%,96.6%and92.3%, compared with IDEXX ELISA Kit on168field serum samples. The Nsp7and N protein specific antibodies levels of piglets vaccinated with PRRSV live vaccine (R98) were detected with Nsp7ELISA kit and IDEXX ELISA kit, respectively. The results showed that the levels of Nsp7antibody in infected groups were positive at14dpi, then gradually rising and lasting more than100days while that of anti-N protein antibody were7days and lasting for70to100days, which both were similar. The PRRSV Nsp7indirect ELISA kit was applied to detect996field serum samples from Anhui, Hebei, Hunan, Jiangsu, Shandong, Sichuan and Zhejiang. The results showed that the seroprevalence was77.21%. The results indicated that the PRRSV Nsp7ELISA kit could be used for PRRSV epidemiology survey and evaluation of vaccine immune effect.In summary, this study has developed a simpler, time-saving, sensitive and specific diagnostic kit for the detection of PRRSV antibodies, which being suitable for large scale monitor of PRRSV infection at low cost and helpful for epidemiological survey of PRRSV and evaluation of vaccine immune effect. |
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