Cloning And Expression Of Mx Gene From Chicken And Identification Of Its Antiviral Specificity

The Mx gene was originally discovered in a genetic line of laboratory mice (A2G) that were influenza resistant, and it was named Mx (myxovirus resistant). Duck Mx protein was found in 1992, chicken Mx gene had yet to be Cloned subsequently. The antiviral specificity of chicken Mx protein is determined by an amino acid substitution at position 631. When it is Asn, Mx has antiviral activities, while it is Ser, Mx has not antiviral activities. So we had a strategy for developing Virus-resistant chickens based on using transgenic techniques, to search for new ideas of enhancement of innate immunity in chicken.A full-length cDNA of chicken Mx gene was obtained using reverse transcription polymerase chain reaction (RT-PCR) amplification of total RNA extracted from chicken embryo fibroblast (CEF) which was induced with Poly(I)?Poly(C). The RT-PCR product was subcloned into pMD19-T Simple vector and the correctness of the sequences was confirmed by sequence analysis, result of sequence analysis showed that its homology with Mx cDNA from GenBank AB088535, AY695797, NM₂04609, XM₄29240, Z23168 was up to 99.6%, 99.5%, 99.4%, 99.7%, 99.4%, at position 2032bp is G.These data indicate that the full-length Mx cDNA was cloned correctly, which will play a great role on next research. We used PCR site-directed mutagenesis technique by which a single amino acid was reciprocally substituted G with A at position 2032bp of chicken Mx cDNA. Sequence analysis confirmed successful mutation from G to A at 2032bp of chicken Mx cDNA. The fragments amplified by PCR containing the mutation site were subcloned into a enteukaryotic expression vector. Then the recombinant vector was transfected into COS-â… cell, Mx gene and Mx protein express were detected by RT-PCR and Indirect Fluorescence Assay (IFA). The results showed that COS-â… cell transfected the recombinant plasmid could stably express the Mx protein. The anti-virus assay showed that Mx protein had characteristics of resistance to infection of Newcastle Disease Virus.By using of the recombinant vector as target exogenous DNA, which was injected to testes of chickens randomly to transfect SSCs, and obtained Transgenic sperm to produce transgenic chickens. The detect of PCR showed that the transgene is stable and can be transmitted to the offspring, and offspring 27.27% (6/22) carried the recombinant vector gene. Our data therefore suggest testis-mediated gene transfer being feasible methods for the generation of transgenic chicken.As a result, we laid a solid foundation and prepared experimental material for the future studies on the Virus-resistant activity of chicken Mx.