Preparation of polyclonal antibody against human MxA protein and its specificity to diversified myxovirus resistant protein A.
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-beta 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-beta induced A549 cells and BL21 proteins expressed with pET32a (+)-MxA. The hybridization signals from IFN-beta induced A549 cells depended on the IFN-beta 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-beta for 48 h showed 95% positive cells. pEGFP-C1-transfected NIH 3T3 cells were all negative.


CONCLUSIONS
Anti-sera are highly specific to diversified MxAs. The antibody is detectable by Western blot, immunocytochemistry and immunofluorescence assay.
Wu
Kang
K
School of Basic Medicine and Biology, Suzhou University, Suzhou 215123, Jiangsu, China.
Liu
Peng
P
Meng
Xiang-Xun
XX
Liu
Li
L
Li
Yan-Hua
YH
Ge
Zheng-Zhen
ZZ
Yang
Ji-Chen
JC
D000818:Animals; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D019204:GTP-Binding Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D064547:Myxovirus Resistance Proteins; D041681:NIH 3T3 Cells; D013045:Species Specificity
C579071:MX1 protein, human; D064547:Myxovirus Resistance Proteins; D019204:GTP-Binding Proteins