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 i...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-CI-wMxA and site-directed mutant MxA pEGFP-Cl-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-[3 induced cells, intact A549, NIH 3T3 cells transfected with pEGFP-CI and pET32a （＋）-transformed BL-21 as controls. Results The antisera had specific positive immunoreactivity to the NIH3T3 cells transformed with pEGFP-CI-wMxA and pEGFP-CI-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-C 1-wMxA and pEGFP-C 1-mMxA had 〉 98% of positive cells at 1：50 dilution of the serum and A549 cells induced by 20 ng/mL IFN-[3 for 48 h showed 95% positive cells. pEGFP-Cl-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.展开更多
基金supported by Educational Committee of Jiangsu Province (Grant No: 07KJD180183)
文摘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-CI-wMxA and site-directed mutant MxA pEGFP-Cl-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-[3 induced cells, intact A549, NIH 3T3 cells transfected with pEGFP-CI and pET32a （＋）-transformed BL-21 as controls. Results The antisera had specific positive immunoreactivity to the NIH3T3 cells transformed with pEGFP-CI-wMxA and pEGFP-CI-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-C 1-wMxA and pEGFP-C 1-mMxA had 〉 98% of positive cells at 1：50 dilution of the serum and A549 cells induced by 20 ng/mL IFN-[3 for 48 h showed 95% positive cells. pEGFP-Cl-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.
