Preliminary Research On Biological Function Of Duck Hepatitis A Virus 2A Protein

【Objective】The nonstructural coding gene of 2A/2A2/2A3 was cloned and inserted into the prokaryotic expression vector p ET-30a(+) to express respectively. The purified proteins as antigen immune the New Zealand white rabbits to prepare the specific polyclonal antibody against 2A, 2A2 and 2A3 of Duck hepatitis virus type A(DHAV). Use double fluorescent report plasmid test 2A1 protein self-cracking function; Use 3C protease activity of cracking experiment to identify whether 2A2 and 2A3 exist alone and verify fully DHAV 2A in the coding region of the genome structure, Besides,the recombinant eukaryotic expression 2A2, 2A3 with GFP fusion protein for exploring the expression and the subcellular localization of 2A2,2A3 in eukaryotic cells.【Methods】(1)The three pairs of specific primers according to the complete genome of duck hepatitis virus A(DHAV)ZJ-V strain(Gen Bank No.EF382778), was designed to amplify 2A, 2A2 and 2A3 gene and inserted into the prokaryotic expression vector p ET-30a(+), construction recombinant expression plasmid p ET-2A, p ET-2A2 and p ET-2A3 were transfected into BL21(DE3) cells, induced by IPTG, the purified proteins as antigen immune rabbits, after three times immune was analyzed with SDS-PAGE and Western blot assay on serum identification.(2)use the eukaryotic expression vector p CI-neo, the recombinant plasmid p CI-GFP-2A-RFP and p EGFP-2A2/2A3 were constructed and transfected into Hela cells. Using fluorescence microscope and protein immunoelectrophoresis test methods, such as 2A1 expression and cracking in the cell and subcellular localization of 2A2 /2A3.(3)According to Fluc N period of coupling a greater than 30 kd protein, its expression greatly was reduced, in view of this,which is together the 3C and 3D DHAV expression and then add cleavage site and fusion fluc on the back, Fluc determined to detect 2A2 between 2A3 SH cracking site whether can be 3C cracking, negative control has nothing to do for a sequence(bp), positive control for the most typical QG DHAV cutting site, and a signal would be introduced in the front with the approval of the GFP gene, rear introduced with membrane localization of RFP genes, inserted between 2A1 gene,recombinant plasmid transfection cell to observe the distribution of two kinds of fluorescence, at the same time set up the insertion of FMDV 2A double fluorescent report gene plasmid as positive control.(4) in PCDNA3.1 DHAV 3C and 3D series expression, in the back to add S/H respectively loci Fluc gene and fusion, different time points after transfection cells.【Results】(1)Using prokaryotic expression vector successfully expressed the 2A,2A2 and 2A3 protein, SDS-PAGE analysis showed 2A protein molecular weight is about 45 ku, 2A2 protein molecular weight is about 25 ku, 2A3 protein molecular weight of about 20 ku, Western blot assay disclosed that the protein could react to the specific positive serum against DHAV indicating that the expression product had well antigenecity. The specific polyclonal antibody against 2A, 2A2 and 2A3 was obtained, when rabbits were immunized with 2A, 2A2 and 2A3 protein.(2)Double fluorescent report plasmid transfection Hela cell can clearly see with contrast consistent results, namely the GFP protein is distributed in the nucleus, and RFP distribution on the cell membrane, protein showed that DHAV 2A1 protein with FMDV and 2A protein in the process of translation is not dependent on other proteins from cracking activity function.(3)transfection luciferase report plasmid were determined Fluc expression capacity after 24 h,36h,48 h,the results show the 3C protease can pyrolysis S/H loci, and cracking efficiency higher than Q/G site.(4)recombinant plasmid p EGFP-2A2 and p EGFP-2A3 transfection eukaryotic cells were determined. The localizationof 2A2 and 2A3 in Hela cells was also determined with focusing microscope, and the result indicated that 2A2 and 2A3 was mainly distributed in the nucleus of Hela cells after 12 h, 24 h, 36 h.【Conclusion】Successfully clone and express the 2A, 2A2, 2A3 gene, the specificity good polyclonal antibody preparation; Implements the 2A, 2A2, 2A3 gene expression in eukaryotic cells and its expression in eukaryotic cells the positioning is analyzed. The gene of DHAV was cloned and expressed both in prokaryoti cand eukaryotic cells, and expressed efficiently in eukaryotic cells, and the subcellular localization of 2A/2A2/2A3 in eukaryotic cells was also first observed. These results are helpful for further study on the structure and function of 2A and the replication mechanism of DHAV.