Establishment Of The Stably Transfected NIH-3T3 Cells With MxA Gene And Analysis Of Its Antiviral Activity To Vesicular Stomatitis Virus(VSV)

the recombination plasmid of pEGFP-C1-MxA encoding the full-length ORF of human MxA(myxovirus resistance protein A)gene was constructed and then pEGFP-C1-MxA/pEGFP-C1 were transfected into NIH-3T3 cells,respectively.The double positive NIH-3T3 cell population for G418 -resistance and expression of EGFP (enhanced green fluorescence protein)/fusion protein of EGFP-MxA were selected and cloned.After attainment of the stably transfected NIH-3T3 cell strains with pEGFP-C1-MxA/pEGFP-C1,these cell population were detected with mice anisera specificity against MxA protein(preparation in our Lab,refer to our other article)by immunoblot/immunocytofluorescence/immunocytochemistry,meanwhile the mRNA of these cell were assayed with the specific primers to MxA sequence by RT-PCR, respectively.Furthermore,these cells were infected with VSV to examine its anti-virus activity.The results show that stressed with 1mg /ml G418,three cell clones (1.4.5,4.3.6,3.2.16)with pEGFP-C1-MxA and one cell clone(EGFP)with pEGFP-C1, which＞98%cells appear enhanced green fluorescence under fluorescence microscope (OLYMPUS IX-71)in 490nm,were obtained by double selection plus 2-3 round monoplast clone.Compared with the EGFP,from samples of the three cell clone(1.4.5, 4.3.6,3.2.16),the specific DNA fragments(about 460bp)can be amplified by RT-PCR and the protein bands in 103kD corresponding to the fusion protein of EGFP-MxA were clearly visualized by immunoblot;and＞98%cells can be recognized by the sera by immunocytofluorescence/immunocytochemistry assay,respectively.Infected with about 50PFU(plague formation Units)/well VSV,No plague was found in two clones and several small plagues was visibled in one clone(1,4,5),while decades of big plagues was revealed in control cell,and Infected with 0.001TCID50 VSV/cell for 48h,Virus yields from three clones(4.3.6,3.2.16,1.4.5)and control clone were 3.5X10~3 and 4.8X10~3 and 5.3X10~5 and 3.5X10~6.From above results,it was indicated that we had obtained two stably transforment cell clones highly expressing MxA protein which have high potent antiviral activity to VSV.