Establishment Of ELISA Based On Recombinant Protein NS1 Of Swine Influenza Virus And Nuclear Localization Of SIV NS And NP Proteins

Swine influenza (SI), caused by swine influenza A virus(SIV), is an acute and contagious respiratory disease of swine. The disease causes great economic loss to pigs industry and threats animal and human health.As a crucial regulator of influenza A virus, nonstructure protein 1(NS1) correlates closely with the virulence of this virus. It displays complex reactions with a variety of cellular proteins, leading to many distinct biological effects. NS1 can also be used to differentiate pigs vaccinated from pigs infected. The NP gene of influenza A is conserved and the nucleoprotein is useful to diagnose and monitor the disease.In this research, the NS1 and NP gene of A/swine/Shanghai/1/2007 were amplified by RT-PCR. The PCR products were cloned into pET-32a(+) and pET-30a(+) vector. The positive recombinant plasmids were identified by PCR and endonuclease digestion. Then recombinant plasmids were sequenced and analysed to make sure the Open Reading Fame(ORF) were correct. The recombinant plasmids pET-32a(+)-NS1 and pET-30a(+)-NP were transformed into BL21(DE3) and BL21(DE3)pLysS competent cells respectively and induced by IPTG with a final concentration of lmmol/L. NS1 and NP proteins were successfully expressed, with the relative molecular weight of about 43.9kD and 63.4kD respectively. The proteins were purified by affinity chromatography and the concentration were determined to be 324.33μg/mL(NS1) and 320μg/mL(NP). Using purified NS1 and NP proteins,6-week-old female Balb/c mice were vaccinated by intraperitoneal injection, and the polyclonal antibody producted and detected by indirect ELISA for NS1 and NP proteins. Using purified recombinant NS1 protein, an indirect ELISA for detection of anti-SIV antibodies was developed and its optimal reaction conditions were determined:the concentration of coating antigen:0.2μg/mL, mice positive serum dilution:1:10000, swine positive serum dilution:1:10, serum reactive time:37℃60min, the final concentration of BL21(DE3) which induced by IPTG and processed by ultrasonicaton:1%, enzyme-antibody dilution:1:10000, enzyme-antibody reactive time:37℃60min, the negative and positive cut-off values:0.286. The ELISA assay was confirmed to have a good specificity, and it can be used to differentiate pigs vaccinated from pigs infected.To study the nuclear localization of SIV NS and NP proteins, eukaryotic expression plasmids encoding NS1 gene, NS gene, NP gene of SIV were constructed, then transfect plasmids into MDCK cells to express the products. The expressed products of pEGFP-N1-NS1, pEGFP-N1-NS, pEGFP-N1-NP were detected by fluorescence microscope after cells stained with DAPI. The expressed products of pCAGGS-NS1, pCAGGS-NS and pCAGGS-NP were detected by immunofluorescence analysis with the produced specific polyclonal antibodies. The expression and intracellular localization of NS1, NS, NP proteins were analyzed and established using a fluorescence microscope. The results show that NS1, NS and NP proteins accumulated in the nucleolus of MDCK cells. To study the nuclear localization of SIV NS and NP proteins was beneficial to research their biological function during the replication of SIV and mechanism of replication.