| Swine influenza is a highly infectious respiratory disease caused by Swine Influenza Virus affecting swine of different stages, sexes and breeds. The clinical signs of Swine Influenza are typical flu-like symptoms. The pigs develop a fever, nasal and ocular discharge, and severe spasms of coughing. They appear lethargic and become anorectic. Swine influenza was first reported in U.S.A in 1918. H1N1 subtype swine influenza wasisolated and identified by Shope in swine in 1930. The disease results economic loss and threats animal husbandry and human health. At present, it widely takes place in many nations and regions in the world. The research of swine influenza has become a hot topic nowadays. Rapid and sensitive diagnosis method is beneficial to control eruption of Swine influenza.In this research, the SIV(H9N2) isolate was propagated in chicken embryos. The virus was purified by ultracentrifuge. According to the structural protein M1,NP and nonstructural NS1 gene sequence published by GenBank, One primer Unit was designed to amplify the cDNA by reverse transcript. The other primers Pm1/ Pm2,Pnp1/ Pnp2,Pns1/ Pns2 were designed to amplify the M1,NP and NS1 gene. The PCR products were cloned into pMD18-T vector and subcloned into pET-28a expression vector. The positive recombinant clone were identified by PCR and endonuclease digest. Then the inserting DNA fragments were sequenced and analysed. The recombinant pET-M1, pET-NP and pET-NS1 plasmids were transformed into BL21 (DE3)/ Rosetta TM(DE3) comptent cell of E.coli and induced by IPTG with a finial concentration of 1 mmo1/L. M1, NP and NS1 gene of the SIV amplified were successfully expressed in Escherichia coli. The recombination protein M1, NP and NS1 with a relative molecular weight of about 31.4,55.1 and 27 kD respectively could be detected by Western-blot, showed good immunogenicity. The results of this study laid a foundation for development of a novel immunological diagnostic method for swine influenza and SIV subunit vaccine.The soluble SIV recombinant nucleoprotein(NP) was expressed by E.coli and purified through native conditions purification method. Using purified soluble nucleoprotein, an indirect ELISA for detection of anti-SIV antibodies was developed and its optimal reaction conditions were determined: coating antigen for 37℃1 hour and 4℃overnight at a concentration of 12.5μg/mL, serum sample (1∶40) and Protein A Peroxidase Conjugated(SPA) being incubated at 37℃for 40 min, the substrate for ELISA being incubated at 37℃for 15 min. The ELISA assay was confirmed to have a good reiteratively, specificity and sensitivity by reiteratively test, cross test and competitive-inhibition test. Performance and optimal cutoff values for the ELISA were determined by using the Two Graph-ROC functions of the CMDT software package, and it was found that the specificity and sensitivity of the ELISA were 95.83% and 91.43% respectively .And compared with the IDEXX Test kit , the agreement rate is about 94%,which showed no significant difference between the two assays. In addition, 356 serum samples collected from swine farms were detected by the developed assay, it was showed that the positive rate was 35.40% for antibody against SIV. The material base was established for assembling test kit for diagnosis of SIV further. Offered a kind of good technological means for diagnosis and monitoring of the disease of SIV. |
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