Cloning And Expression Of MxA Gene From Chickens In E.coli And Identification Of Biologica Activity Of MxA

MxA was a new kind of antivirus protein that was induced by IFNα/β. Mx genes are found in a variety of organisms, including yeast and vertebrates ranging from fish to humans. Mx proteins are GTPase enzymes induced by interferon (IFN). The MxA proteins contain putative tripartite GTP-binding sites as well as a leucine zipper with two elements. The cytoplasmic human MxA protein confers resistance to influenza virus, VSV, measles virus, and Thogoto virus. However, cytoplasmic human MxB and rat Mx3 are without antiviral activity. In addition, almost all laboratory mouse strains carry nonfunctional Mx1 and Mx2 genes, but feral mouse strains contain functional antiviral Mx1 and Mx2 proteins. In the duck, Mx protein found in the nucleus and cytoplasm showed no enhanced influenza virus resistance. On the basis of the comparison among the antiviral activities associated with many Mx variations, a specific amino acid substitution at position 631 (Ser to Asn) was considered to determine the antivirally positive or negative Mx gene.When it is Asn,MxA has antiviral activities; while it is Ser, MxA has not antiviral activities.The complete nucleotide sequence of chicken Mx cDNA and the function of enhanced resistance to viruses were reported earlier. In this study, one of specific primer for chicken MxA cDNA was designed and synthesized according to the previously published gene sequence of MxA. The full length cDNA gene of MxA was amplified from Poly:IC and interferon (IFN) CEF cells by Reverse Transcription-Polymerase Chain Reaction(RT-PCR). The it was cloned into pMD18-T vector.To highly express chicken MxA in E.coli and purify the fusion protein, cDNA fragment encoding the MxA was subcloned into the downstream of GST gene according to the right open reading frame(ORF) in the prokaryotic expression vector pGEX-6P-1. The recombinant plasmid was transformed into E.coli DH5αstrain, and then induced by IPTG at 37℃. The recombinant MxA was expressed efficiently in forms of inclusion body with the yield accounting for 30% total bacteria protein. Optimizing select the parameters of expression, the level of expression of MxA is highest when IPTG reachers...