| Transmissible gastroenteritis virus (TGEV) and porcine epiemic diarrhea virus (PEDV) which blong to the members of the family Coronaviridae are major etiological agents of diarrhea and death in piglets. These diseases were happened all over the world and causes great harm to pig industry. In the present study, double RT-PCR and double real-time reverse transcriptase (RT)-PCR were developed for diagnosing PEDV and TGEV; At the same time, the prokaryotic expression vector pET28a(+)-P-SP1 was constructed and S protein of PEDV was expressed, Furthermore, the immune efficacy of the recombinant S protein was investigated. The most research works were as following:Based on the gene sequences of porcine epidemic diarrhea virus (PEDV) CV777 strain and transmissible gastroenteritis virus (TGEV) Purdue P115 strain reported in GenBank, a specificity upper primer was designed in consensus sequence of PEDV and TGEV, and two down primers for PEDV and TGEV, respectively. The PCR products of PEDV and TGEV had molecular sizes of 213bp and 546bp, respectively. The sensitivity test and specifity test results showed that 102copies/μL cDNA can be detected by the double RT-PCR and no reations could be found. It indicated that the sensitivity and specificity of the double RT-PCR was good through the primary application. The result of the research supplies a reference method to diagnosis for the two diseases.According to PEDV-N gene sequence available in GenBank, the specific primers and TaqMan probe were designed by using Beacon Designer software. A 186 bp fragment can be amplified with the primers. With standard DNA obtained from the plasmid cloned with PEDV-N gene, a real-time quantitative PCR for PEDV was established successfully. The sensitivity of the assay was 10copies/μL plasmid DNA, assay. The assay also proven to be specific based on the results that no amplification was found by detecting RNA/DNA from PCV, JEV, SFV, PRRSV and PPV. The coefficient variation (CV) of intra/inter-assay for same DNA sample was less than 2%. The results indicated that the standard curves were shown to be of high linearity, specificity, sensitivity and reproducibility. Based on the principles of TaqMan probe quantitative assay, the two pairs of primers and two quantitive TaqMan probes which were tagged with FAM and JOE were synthesized respectively. Double real-time reverse transcriptase (RT)-PCR was developed for diagnosing PEDV and TGEV by the optimization of reaction conditions. The method possessed high sensitivity, reproducibility and specificity. The minimum of detection was 2×101 copies/μL RNA. The variabilites of the intra-assay and inter-assay were evaluated with standard solutions of each transcript were less than 2% of coefficients variation (CV). The assay was also proven to be specific based on the results that no amplification was found by detecting RNA/DNA from PCV, JEV, SFV, PRRSV, PRV and PPV.Based on the gene sequence of porcine epidemic diarrhea virus (PEDV) CV777 strain S gen reported in GenBank, the specificity primers were designed. The purpose gene of porcine epidemic diarrhea virus CH/ZJ strain was amplified by RT-PCR. The amplified DNA fragment was cloned into prokaryotic expression vector pET28a(+) and sequenced. The recombinant plasmid was transformed into E.coli BL21-DL3 (Rosetta) and induced with IPTG. Thin-layer scanning showed that the expression product accounted for 80% of the total bacterial proteins. The amount of expressed protein reached peak at 2.5 hours after the induction. The results of SDS-PAGE and Western blotting showed that the protein was 36.7 ku in size and specifically reacted with PEDV positive sera from pigs, but didn't with the negative sera. These results lay the foundation of studying novel vaccine and developing effective diagnostic method. |
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