| Porcine deltacoronavirus(PDCo V)is a new emerging coronavirus,and there are few diagnostic methods for PDCo V Accessory proteins are genus-specific for coronavirus.the NS6 is one putative accessory proteins encoded by PDCo V.It is reported that many auxiliary proteins participate in the immune regulation in the body and affect the pathogenesis of the virus.In this study,The Ns6 gene of PDCo V Sichuan strain was cloned and the bioinformatics analysis was conduceted.The prokaryotic expression of PDCo V Ns6 recombinant protein was used as the coated antigen,and an indirect ELISA antibody detection method of PDCo V was established and evaluated by detecting clinical samples.1.Cloning and bioinformatics analysis of PDCo V Ns6 gene.The primers were designed according to the Ns6 gene sequence released by NCBI,and the Ns6 gene of Sichuan strain PDCo V-SC2015 was amplified by RT-PCR.The gene sequencing and mutation analysis was subsequently performed The results showed that Ns6 gene was 285 bp in length,it has 99%gene similarity comparing with Ns6 gene of CH/Sichuan/S27/2012 strain.The amplified PDCo V Ns6 gene was named as CHN-SC2015strain Ns6 gene,compared with CH/Sichuan/S27/201 strain,mutations occurred in the nucleotides.Phylogenetic analysis showed that the Ns6 gene of CHN-SC2015 strain was close to those NS6 genes of the PDCo V strains isolated from the China's mainland.The gene was relatively close to the CH/Sichuan/S27/2012 strain,HKU15/S579 strain,CH/SXD1/2015 strain and CHN-LYG-2014 strain and far from the evolution of American and Korean strains.The predicted amino acid sequence of the Ns6 protein of the CHN-SC2015 strain showed that the protein was an unstable hydrophilic protein without glycosylation site.The secondary structure prediction showed that there was abundant?-helix in the protein and no transmembrane region exist in the protein.2.Prokaryotic expression of PDCo V Ns6 protein and preparation of rabbit anti-NS6protein polyclonal antibody.The recombinant protein of Ns6 was expressed by p ET-32a(+)vector,The recombinant protein was 32 k Da in weight.The optimal expression condition was inducing the Transsetta strain under the final concentration of IPTG 0.6 m M 5 h on 30?.The infusion analysis showed that the recombinant protein was expressed in supernatant.After Ni+affinity chromatography method and the ultrafiltration tube concentration,the 1.2 mg/m L purified protein was obtained.A mixture of purified Ns6 protein and Freund's adjuvant immunized New Zealand rabbits 3 times and the titer of the purified antibody was determined by indirect ELISA at a level of above 10~5.The rabbit anti-ns6 protein polyclonal antibody could be used for the study of the PDCo V Ns6 protein.3.PDCo V Ns6 protein indirect ELISA detection method was established.In order to provided a serological diagnostic tool for PDCo V,an indirect ELISA,r PDCo V-Ns6-ELISA,which used rocombinant NS6 protein as coating antigen.In this study r PDCo V-Ns6-ELISA was assessed by testing optimum reaction conditions,determining the cut-off value,specificity and repetition.The results indicated that the optimum antigen coating-concentration is 100?g/m L,serum dilution is 1:100,concentration of anti-pig Ig G peroxidase conjugate was 1:5000,the reaction time of samples was 30 min and the best rcation time of anti-pig Ig G peroxidase conjugate was 1h.The recombinant protein antigen Ns6 had no cross-reaction with antisera against other five swine viruses Sensitivity of NS6-ELISA was 1:6400 when the cut-off value was 0.43.The coefficient of variability percent(C/V%)of intro-batch duplicativity test and inter-batch duplicativity test was less10%.A total of 387 serum samples collected in Sichuan area were detected by NS6-ELISA,the positive rate of PDCo V was 0.52%.The Ns6-ELISA has good sensitivity and specificity,which can be used for detecting PDCo V antibodies. |
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