Expression Of Human Norovirus NS1/2 Protein And Its Relationship With Interferon Response

Objectives Gene recombination technique was conducted to express human norovirus(Hu NV)nonstructual(NS)1/2 protein in vitro,furthermore to explore the key sites of Hu NV NS1/2 protein cleavage.Finally,the effect of Hu NV NS1/2 protein on interferons(IFNs)secretion of host cell was studied.Methods NS1/2 protein sequence of different Hu NV types was downloaded from National Center for Biotechnology Information thereafter the nucleotide sequence was synthesized.Digested by Bam HI and Xho I,and linked to p CAGGS,p VR1012 vector to express in vitro through eukaryotic protein expression system,and linked to pc DNA3.1 vector for eukaryotic and in vitro transcription and translation system expression,linked to p ET30 a to express in prokaryotic protein expression system,Coomassie brilliant blue staining and western blot were used to verify NS1/2 protein expression and cleavage.Protein mass spectrometry was performed to analyze truncated protein peptide sequence and truncated protein.Screen the potential cleavage region of NS1/2 protein by size and other methods,determine the key cleavage site by combining site-directed mutagenesis and western blot,analyze possible proteases through bioinformatics tools,and verify it by protease activator/inhibitor.The dual fluorescent reporter gene system and quantitative Real-time PCR(q PCR)were used to verify the effect of NS1/2 protein on the expression level of IFNs.Results 1 NS1/2 protein was successfully expressed in the prokaryotic protein expression system,but the bands were messy and the expression level was very low;the eukaryotic protein expression system could successfully express the NS1/2 protein and the truncated protein after being lysed by the host cell could be observed.Rabbit reticulocytes successfully translated NS1/2 protein in vitro,but no truncated protein was observed;insect expression system can express NS1/2 protein but the expression is very low.2 GII.4 Sydney [P16],GII.4 Sydney [P4 New Orleans],Norwalk type Hu NV NS1/2 protein can be expressed and cleavaged in HEK 293 T cells;the key cleavage site of GII.4 Sydney [P16] NS1/2 is located at the 289 th amino acid;4 GII.4 Sydney [P16] NS1/2 cleavage is not affected by cysteinyl aspartate specific proteinase(Caspase).The effect of activator/inhibitor,bioinformatics tool analysis may show that cathepsin G enzyme plays a lytic effect.5 GII.4 Sydney [P16] NS1/2can inhibit the expression of IFN-?/? in Human embryonic kidney(HEK)293T cells.Conclusions 1 GII.4 Sydney [P16],GII.4 Sydney [P4 New Orleans],Norwalk type Hu NV NS1/2 proteins can be expressed and cleavaged in eukaryotic cells;2 The key cleavage site of GII.4 Sydney [P16] NS1/2 is located at the 289 th amino acid and has little effect by Caspase family members;3 GII.4 Sydney [P16] NS1/2 can inhibit the expression of IFN-?/?in HEK 293 T cells.Figures 39;Tables 18;References 109...