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-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.

Expression of MxA Protein in Triple Negative Breast Cancer and its Relationship with Prognosis

Objective:To explore the expression of human myxovirus resistance protein A(MxA)in triple negative breast cancer(TNBC)and its relationship with prognosis.Methods:144 cases of TNBC confirmed by pathology before or after surgery from January 2014 to January 2017 in the First Central Hospital of Baoding City were retrospectively collected.Western blotting was used to detect the expression of MxA protein in TNBC and adjacent breast tissues.According to the expression of MxA protein,144 TNBC patients were divided into low MxA protein expression group(n=91)and MxA protein high expression group(n=53)for subsequent comparison of prognosis of patients in between these two groups.Results:The expression of MxA protein in TNBC tissue was lower than that of adjacent breast tissue,and the difference was statistically significant(P<0.05).The patients in high MxA expression group had higher loco-regional recurrence-free rate,disease-free survival(DFS)rate,and overall survival(OS)rate than those in low MxA expression group for 3 years.On the other hand,the distant metastasis rate was lower in the high expression group compared to that in the low MxA expression group,and the difference was statistically significant(P<0.05).Conclusion:In triple-negative breast cancer,high MxA expression is a potential predictor of TNBC prognosis.

GTPase Activity of MxB Contributes to Its Nuclear Location, Interaction with Nucleoporins and Anti-HIV-1 Activity

The human myxovirus resistance 2(Mx2/Mx B)protein,a member of interferon(IFN)-inducible dynamin-like large GTPases,restricts a number of virus infections.Inhibition of these viruses occurs at poorly-defined steps after viral entry and has a common requirement for Mx B oligomerization.However,the GTPase activity is essential for the anti-viral effects of Mx B against herpesviruses and HBV but not HIV-1.To understand the role of Mx B GTPase activity,including GTP binding and GTP hydrolysis,in restriction of HIV-1 infection,we genetically separated these two functions and evaluated their contributions to restriction.We found that both the GTP binding and hydrolysis function of Mx B involved in the restriction of HIV-1 replication.The GTPase activity of Mx B contributed to its nuclear location,interaction with nucleoporins(NUPs)and HIV-1 capsids.Furthermore,Mx B disrupted the association between NUPs and HIV-1 cores dependently upon its GTPase activity.The function of GTPase activity was therefore multi-faceted,led to fundamentally distinct mechanisms employed by wild-type Mx B and GTPase activity defective Mx B mutations to restrict HIV-1 replication.