The Study Of The Antiviral Activity Of Gallus Mx On NDV

Interferon can induce three well-known antiviral pathways-Mx, OAS, PKR. Mx gene was found by Lindenmann and acquired from mouse chromosome 16. It was named Mx(Myxovirus-resistant) gene due to its ability of resisting Myxovirus.Mx proteins belong to the dynamin superfamily of large GTPase, and are induced by type I interferons in vivo. Most vertebrates possess one to three Mx isomers which locate in either nucleus or cytoplasm. Mx proteins can resist a variety of viruses including both RNA and DNA virus, for instance, Influenza virus, VSV, Thogoto virus, Hantaviruses, Rabies virus, HBV, and so forth. At present, the research of Mx gene mainly focuses on human MxA and mouse Mx1; however, the study on gallus Mx gene is so limited and there is no report about gallus Mx resisting Newcastle disease virus (NDV) which is the virus that can infect and kill birds.Constructing and screening cell line that stably expressed Mx protein are basis of studying the antiviral activity and mechanism of Mx protein. The RNA of chicken was extracted from chichen blood and used as the template for RT-PCR to obtain the cDNA. The cDNA was used as the template in PCR to amplify gallus Mx gene, and the acquired fragment was cloned into the pCI-neo vector. The plasmid was transfected into Swiss 3T3 cells. For screening the cell line that stably expressed gallus Mx protein, RT-PCR, Western blotting and IFA were utilized.Crystal violet dying and real time PCR were used to prove the antiviral activity of gallus Mx protein by using Swiss 3T3-Mx cell line.NP is very crucial to packaging of virion of NDV. This work primarily showed the possible mechanism of gallus Mx resisting NDV was due to the interaction between Mx protein and NP, however, the exact mechanism was not clearly proved.In order to investigating the mechanism that gallus Mx protein inhibited NDV, the interaction between gallus Mx protein and the certain component of NDV was studied by using co-IP, IFA and mass spectrum. Because of the same size between NP and large fragment of antibody, the interaction between gallus Mx protein and NP can not be proved. To some extent, the result of co-IFA illuminated overlap between Mx protein and NP. And the result of MS was not got yet. It is possible that the interaction between gallus Mx protein and NDV NP inhibits the packaging of virion, and further investigation is needed. For investigating human MxA protein, MxA gene was cloned, and MxA protein was expressed in E.coli by use of pET32a vector. The result of SDS-PAGE showed high expression of the target protein. Purified MxA protein was used to prepare monoclonal antibody in mice. ELISA showed this monoclonal antibody can bind to MxA protein specificly, and Western blotting proved this antibody can be used in testing expression of MxA protein in human cells.In conclusion we constructed and screened Swiss 3T3-Mx cell line that stably expressed gallus Mx protein and proved antiviral capability of gallus Mx resisting NDV for the first time. It is possible that the interaction between gallus Mx protein and NP resulted in this antiviral activity. Meanwhile, the anti-MxA monoclonal antibody was prepared and this antibody can be used to detect MxA protein.