In vitro Interaction Analysis Between Muscovy duck parvovirus Non-structural Protein NS1 and Waterfowl Eukaryotic Translation Elongation Factor 1A1

doi:10.7668/hbnxb.20192150

Abstract

Abstract: This study was conducted to investigate the interaction relation between Muscovy duck parvovirus non-structural protein NS1 and waterfowl eukaryotic translation elongation factor 1A1(eEF1A1). According to the published eEF1A1 gene sequence of Pekin duck and Zhedong white goose in GenBank, the artificially synthesized plasmids were named pUC57-DeEF1A1 and pUC57-GeEF1A1 respectively after optimization of the preferred codons of E. coli;The recombinant plasmids pUC57-NS1, pUC57-DeEF1A1 and pUC57-GeEF1A1 containing the NS1 gene of MDPV YL08 strain were digested with Bam HⅠand Xho Ⅰ, and the target fragments were recovered and inserted into the prokaryotic expression vectors pGEX-6p-1 and pET-32a to construct recombinant plasmids pGEX-DeEF1A1, pET-GeEF1A1, pET-NS1 and pGEX-NS1, respectively;The recombinant plasmids were transformed into E. coli Rosetta(DE3), and induced by IPTG, the expression of recombinant proteins GST-DeEF1A1, TRX-GeEF1A1, TRX-NS1 and GST-NS1 were obtained;The HRP-labeled GST monoclonal antibody was used to identify GST-DeEF1A1 and the HRP-labeled His monoclonal antibody was used to identify TRX-GeEF1A1;The HRP-labeled His monoclonal antibody was used to identify TRX-NS1 and the HRP-labeled GST monoclonal antibody was used toidentify GST-NS1, and the MDPV infected Muscovy duck serum was used to identify the antigenicity of TRX-NS1 and GST-NS1;GST pull-down test was used to verify the interaction between GST-DeEF1A1 and TRX-NS1, GST-NS1 and TRX-GeEF1A1. The results showed that the molecular weights of GST-DeEF1A1, TRX-GeEF1A1, TRX-NS1 and GST-NS1 were 76.9, 67.9, 89.5, 98.6 ku, respectively;GST-DeEF1A1 and GST-NS1 could specifically bind to HRP-labeled GST monoclonal antibody, while TRX-GeEF1A1 and TRX-NS1 could specifically bind to HRP-labeled His monoclonal antibody;GST-NS1 and TRX-NS1 could specifically bind to MDPV infected duck serum with good antigenicity;In GST pull-down test, GST-DeEF1A1 could interact with TRX-NS1, but GST-NS1 could not interact with TRX-GeEF1A1. This results showed that MDPV NS1 protein could interact with Pekin duck eEF1A1 in vitro but not with Zhedong white goose eEF1A1 in vitro and the interaction difference might be related to the 102-106 aa and 108 aa amino acid residues.

Key words: Muscovy duck parvovirus, Non-structural protein, Waterfowl eukaryotic translation elongation factor, GST pull-down, Protein interaction

CLC Number:

S432.4
Q78

YU Tianfei, XIE Pengyu, SUN Wanshu, LI Jing, YIN Haichang, LI Ming, YU Zhidan. In vitro Interaction Analysis Between Muscovy duck parvovirus Non-structural Protein NS1 and Waterfowl Eukaryotic Translation Elongation Factor 1A1[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(4): 232-238. doi: 10.7668/hbnxb.20192150.

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