Preparation of Polyclonal Antibody against Human MxA Protein and Its Specificity to Diversified Myxovirus Resistant Protein A

摘要

Objective To study the human myxovirus resistant protein A(MxA),a specifically induced peptide by interferon I,and to use its level as a diagnostic criterion for viral infections.Methods Anti-MxA antisera from immunized mice were prepared with the expressed MxA protein of pET32a-MxA in E.coli BL-21(DE3).To confirm the antiserum activity and specificity,the expression product of BL21,wild type MxA pEGFP-C1-wMxA and site-directed mutant MxA pEGFP-C1-mMxA(N589S)stably transfected 3T3 cells and induced A549 cells were detected by Western blot with the antisera using non-MxA transfected or non-IFN-β induced cells,intact A549,NIH 3T3 cells transfected with pEGFP-C1 and pET32a(+)-transformed BL-21 as controls.Results The antisera had specific positive immunoreactivity to the NIH3T3 cells transformed with pEGFP-C1-wMxA and pEGFP-C1-mMxA,INF-β induced A549 cells and BL21 proteins expressed with pET32a(+)-MxA.The hybridization signals from IFN-β induced A549 cells depended on the IFN-β inducing concentrations.Meanwhile,immunohistochemical assay showed that NIH 3T3 cells with pEGFP-C1-wMxA and pEGFP-C1-mMxA had > 98% of positive cells at 1:50 dilution of the serum and A549 cells induced by 20 ng/mL IFN-β for 48 h showed 95% positive cells.pEGFP-C1-transfected NIH 3T3 cells were all negative.Conclusion Anti-sera are highly specific to diversified MxAs.The antibody is detectable by Western blot,immunocytochemistry and immunofluorescence assay.