志贺氏杆菌分泌蛋白Dnak的表达纯化与多克隆抗体的制备及应用OA北大核心CSTPCD

The purpose of this study was to express the secreted Shigella protein Dnak(Hsp70)in rotavirus-infected suck-ling mice,and to prepare Dnak polyclonal antibodies.The extracted Shigella genome was used as a template to amplify the Dnak fragment by PCR.The prokaryotic expression plasmid pET24a(+)-Dnak and eukaryotic expression plasmid PCDNA3.1-Dnak were ligated with pET-24a(+)and PCDNA3.1,respectively,and the pET24a(+)-Dnak prokaryotic expression plasmid was transformed into E.coli BL21(DE3)competent cells.After IPTG-induced expression,Dnak protein was purified with a nickel column.Polyclonal antibodies were prepared from the purified recombinant protein for immunogenic C57BL/6 mice,and the antibody titer was detected by indirect ELISA.Antibody sensitivity and specificity were detected by western blotting.The interaction between Dnak and rotavirus non-structural protein Nsp2 was detected by GST pull-down assay.The eu-karyotic expression plasmid PCDNA3.1-Dnak was transfected into HEK293T cells,and the prepared Dnak polyclonal anti-bodies were used to detect the expression of Dnak in cells by western blotting.MEGA11 and Genedoc software were used to an-alyze the amino acid sequence homology between Shigella and other bacteria of the genus Shigella.The recombinant plasmids pET24a(+)-Dnak and PCDNA3.1-Dnak were sequenced and found to contain genes consistent with the sequence of Shigella:PRJNA804371,thus demonstrating successful plasmid construction.The induced expression of recombinant Dnak protein re-sulted primarily in soluble forms with a relative molecular weight of approximately 75 kDa;a concentration of 0.62 mg/ml;and good reactivity and immunogenicity.Dnak mouse polyclonal antibodies reacted specifically with recombinant Dnak protein at a titer of 1:32 000,and the limit of Dnak protein detection was a dilution of 1:6 000,thus indicating high titer and sensitivity to meet experimental needs.GST Pull-down assay can detect the interaction of Dnak with the rotavirus non-structural protein Nsp2.HEK293T cells transfected with PCDNA3.1-Dnak recombinant plasmid expressed Dnak protein.The PRJNA804371 strains were compared with MEGA11 and Genedoc software,and the amino acid sequence homology between the strains and Shigella sonnei,Shigella dysenteriae,Shigella boydii and Shigella flexneri was high.