ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Anti-sera are highly specific to diversified MxAs. The antibody is detectable by Western blot, immunocytochemistry and immunofluorescence assay.</p>
Subject(s)
Animals , Humans , Mice , Antibody Specificity , Cell Line, Tumor , GTP-Binding Proteins , Genetics , Allergy and Immunology , Metabolism , Gene Expression Regulation , Myxovirus Resistance Proteins , NIH 3T3 Cells , Species SpecificityABSTRACT
Objective To prepare polyclonal antibodies against human PIWIL and to identify their property and tissue distribution of PIWIL.Methods PIWIL polypeptide was synthesized and conjugated to Keyhole limpet hemocyanin(KLH) as an immunogen.Then PIWIL-KLH conjugations were injected into rabbits subcutaneously to produce polyclonal antibodies.The specificity and sensitivity of antibodies were identified by ELISA and Western blot after purification by affinity chromatography.PIWIL were then stained on the tissue chip to study their distribution.Results Rabbit antibodies against PIWIL were prepared after injection of PIWIL-KLH conjugation.These antibodies specially recognized PIWIL peptides.Expression of PIWIL was found in the cytoplasm of epithelia cells of varied normal tissues and tumor tissues.Conclusion The successful preparation of the polyclonal antibody against PIWIL will provide an efficient reagent for further study of its role in the pathway of miRNA and RNA interference and in the pathogenesis of human disease.
ABSTRACT
Objective:To prepare rabbit polyclonal antibody against human argonaute 3(AGO3) protein,to identify its properties and investigate the tissue distribution of AGO3 using tissue array.Methods:AGO3 peptide was synthesized using chemical method,and then conjugated to Keyhole limpet hemocyanin(KLH) as immunogen.The AGO3-KLH conjugate were injected into rabbits subcutaneously to produce polyclonal antibodies.The specificity and sensitivity of antibodies were identified by ELISA and Western blot after purification using affinity chromatography.Then the distribution of AGO3 in tissues was examined through immuno-stainning by tissue array.Results:Rabbit polyclonal antibodies against AGO3 were raised after immunization with AGO3-KLH conjugates.The anti-serum titer after the last inoculation was up to 1∶20 000.The preparations of the antibody were confirmed to raised recognize AGO3 peptides specially by ELISA and Western blot.AGO3 protein was stained positively in the cytoplasm of tumor cells and epithelial cells in many normal tissues.Conclusion:The polyclonal antibody against AGO3 protein has been achieved successfully,and it provides an efficient tool for further studying the roles of AGO3 in the pathway of miRNA and RNA interference and in the pathogenesis of human disease.