Article: Cloning and production of nucleoprotein of swine influenza virus in E.coli
Abstract: Influenza A virus is a causative agent of swine influenza which is an acute respiratory disease affecting pigs at all ages leading to considerable economic losses of swine industries. Monitoring and surveillance of swine influenza virus (SIV) infection ... ...
| Abstract | Influenza A virus is a causative agent of swine influenza which is an acute respiratory disease affecting pigs at all ages leading to considerable economic losses of swine industries. Monitoring and surveillance of swine influenza virus (SIV) infection and herd immunity are essential for disease prevention and control. However, it requires an effective diagnostic tool such as ELISA. Nucleoprotein (NP) is a major group specific and conserved protein suitable to be use as antigen for the detection of all subtypes of influenza A viruses. The objective of this study was to production and purification of the recombinant NP protein. Total RNA was extracted from swine influenza virus (A/swine/IA/1930) suspension. cDNA was synthesis from the total RNA and NP gene was amplified using NP specific primers which designed to contain KpnI and SalI at 5 ends of the forward and reverse primers, respectively. The NP gene was ligated to the vector, pQE80L, prior to be transformed into E.coli strain DH5 alpha. In E.coli. The expressed protein reached the highest yield when the expression was induced using 0.2 mM IPTG at 37 deg C for 24 hours. Majority of the protein was insoluble and accumulated as inclusion bodies. The insoluble NP protein was dissolved in 8 M Urea pH 8.0 and purified using a nickel-charged resin-affinity column. The protein was then neutralized using stepwise dilution method. The recombinant protein had a molecular weight approximately 56 kDa and reacted strongly to convalescent serum from a pig infected with SIV. The results showed that the recombinant NP protein was successfully produced. It is immunogenic and ready for further diagnostic application |
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| Keywords | Swine ; Viruses ; Animal diseases ; Viroses ; Influenzavirus ; Molecular cloning ; Genetic engineering ; Animal production ; Nucleoproteins |
| Language | Thai |
| Document type | Article |
| Database | AGRIS - International Information System for the Agricultural Sciences and Technology |
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