[Prokaryotic expression and identification of human astrovirus nonstructural proteins, nsP1a and nsP1a/4]

Human astrovirus (HastV) is recognized as one of the leading causes of acute viral diarrhea in infants. The HastV non-structural protein, nsPla, and C-terminal protein, nsPla/4, contain various conserved functional domains,and may play an important role in virus replication, transcription and the virus-host interactions of HastV. This study used an E. coli system to investigate the expression of nsPla and nsPla/4 proteins. Firstly,the nsPla and nsPla/4 genes of HAstV-1 were cloned into the prokaryotic expression vector,PGEX-4T-1, to build the PGEX-4T-1a and PGEX-4T-la/4 fusion protein plasmids. Then, the recombinant plasmids were transformed into Escherichia coli BL21 (DE3) and induced with isopropyl-β-D-thiogalactopyranoside (IPTG). The optimal expression conditions of the two fusion proteins were identified and then analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting, respectively. The results showed that the pGEX-4T-la fusion protein was maximally expressed at 30 °C after 12 hours of induction with 1.0 mM IPTG. The pGEX-4T-la/4 fusion protein was maximally expressed at 20 °C after 8 hours of induction with 0.5 mM IPTG. Western blot analysis showed that the two fusion proteins specificity reacted with the anti-nsPla and anti-GST monoclonal antibodies, respectively. This study successfully obtained the HAstV non-structural protein, nsP1a, and its C-terminal protein nsP1a/4 protein using an E. coli system. This novel study lays the foundation for future research into the pathogenic mechanisms of human astrovirus and the functions of its non-structural protein.