[Preparation and characterization of polyclonal antibody against Ia-associated invariant chain of Muscovy Duck (Cairina moschata)].

OBJECTIVE:To prepare polyclonal antibody against invariant chain of Muscovy duck (Cairina moschata) (MDIi) and identify its reaction with MDIi extracted from tissues of Muscovy duck. METHODS:MDIi was amplified by PCR and used to construct the prokaryotic expression vector of pET-32a/MDIi by linking with the plasmid of pET-32a. Then pET-32a/MDIi was transformed into E.coli Rosetta to induce the prokaryotic expression. After identified by SDS-PAGE, prokaryotic expression products were further purified from running gel of SDS-PAGE and injected into mice to prepare polyclonal antibody against MDIi. The titer and specificity of the polyclonal antibody against MDIi were analyzed by indirect ELISA and Western blotting, respectively. The intensity of reaction between the polyclonal antibody and MDIi extracted from tissues of Muscovy duck was also identified by indirect ELISA. RESULTS:The prokaryotic expression vector pET-32a/MDIi was successfully constructed. About 40 kD recombinant proteins of MDIi were confirmed to be expressed in the form of inclusion body in Rosetta. Polyclonal antibody against MDIi with a titer of 1:128 000 was obtained from the immunized mice and its high specificity was demonstrated by Western blotting. The titer of reaction between the polyclonal antibody and MDIi was 1:32 000. CONCLUSION:The polyclonal antibody against MDIi was successfully prepared with a high titer and specificity. It has a strong immune reaction with MDIi extracted from tissues of Muscovy duck.