Prokaryotic expression and polyclonal antibody preparation of human adenovirus type 7 DNA binding protein / 中华预防医学杂志

ABSTRACT

Objective: To express DNA-binding protein (DBP) of human adenovirus (HAdV) type 7 using the prokaryotic expression system, and product anti-HAdV-7 DBP rabbit polyclonal antibody. Methods: The HAdV-7 DBP gene was synthesized and cloned into prokaryotic expressing vector pET30a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cell. The recombinant protein DBP was expressed by induced Isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified with Ni-NTA affinity column. The titer of anti-DBP polyclonal antibody produced in immunized rabbit was measured by indirect ELISA, and the specificity of the antibody was identified by Western blotting and indirect immunofluorescence assay (IFA). In addition, purified rDBP was used as coating antigen for indirect ELISA assay to detect specific IgM and IgG antibodies against DBP in the serum of children infected with HAdV. Results: The HAdV-7 DBP plasmid was constructed successfully. The purified recombinant DBP was more than 95% after purification. The titer of polyclonal antibody was 1∶1 024 000. The polyclonal antibody showed high specificity in vitro using Western blotting and IFA. The positive rate of specific anti-DBP IgM and IgG antibody in acute-phase serum samples collected from children infected with HAdV were 50.0% (19/38) and 63.2% (24/38), respectively, using indirect ELISA. Conclusion: In summary, the HAdV-7 rDBP is expressed using prokaryotic expression system, and the recombinant HAdV-7 DBP protein and the anti-DBP rabbit polyclonal antibody with high titer are prepared.