Develoment of ELISA for detecting antibody against influenza A nucleoprotein in pigs

Abstract

Serological diagnosis is the major role in the surveillance of influenza A virus occurrence in animal herds including pig herd. Commercial ELISA is a convenient and practical method, but it is expensive for routine work in Thailand. The objective of this study is to produce a recombinant nucleoprotein of swine influenza virus for developing an ELISA-based test to detect antibody against nucleoprotein (NP) in order to use it as influenza A seroscreening test in Thai pig herds. Viral RNA was extracted from swine influenza H1N1virus (A/swine/Thailand/CU-CBP18/2009). The full-length NP gene (1,500 bp) and truncated NP gene (1,000 bp) were amplified by RT-PCR. These RT-PCR products were ligated into pThioHisA E.coli expression vector. The recombinant NP-pThioHisA and NPt-pThioHisA E. coli expression vectors were constructed and transformed into host cells (E. coli strain TOP10). The recombinant full-length NP and truncated NP proteins were expressed by using 1 mM of IPTG. Our result showed that the expressed full-length NP and truncated NP proteins were detected by SDS-PAGE (56 kDa and 48 kDa, respectively). Moreover, these proteins could be detected by commercial ELISA using for detection of IAV NP antigen. However, these NP proteins were expressed in insoluble forms, so the proteins could not be purified in native condition. In order to solve this problem, various optimizing conditions were used for enhancing the solubility of the expressed proteins, including the increasing of incubation period in denaturant (6 M guanidide hydrochloride) and using variation of protein expression conditions, including induction temperature, concentration of IPTG, and bacterial strains. Our results demonstrated that, the expressed NP proteins were not purifiable, leading to the unsuccessful establishment of the ELISA-based method. However, this research can be used as the basic information for the future researches that need to produce the recombinant NP proteins for developing an ELISA test kit for antibody detection against NP protein of influenza A virus.