Study On The Polymorphism Of Mx Gene In Chicken And Location Of Mx Protein In Cells

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 polymorphism of Mx gene and developing Virus-resistant chickens based on using transgenic techniques, to search for new ideas of enhancement of innate immunity in chicken.This study with a mismatch PCR-restriction fragment length polymorphism (PCR-RFLP) methods on 15 breeds of chicken Mx gene detection. Aim is detection the difference of frequency of Mx resistance gene (A) and susceptibility gene (G) in different breeds. The results showed that 14 breeds of chicken Mx gene 2,032 points exist two gene alleles, three kinds of genotype, which Luyuan chicken, Langshan chicken, cock fight chicken found no AA genotype, Chahua chicken found no GG genotype; the average of resistance gene (A) and sensitive gene (G) is 0.358, 0.642. The frequency of resistance alleles A is from 0.034 to 0.846. Meanwhile the result of Chi Square test indicated that all the populations were fit with Hardy-Weinberg equilibrium.Designed primers of EGFP reporter gene, meanwhile removed the stop codon. At the same time joined the restriction sites at both ends. We used high-fidelity enzyme to amplify EGFP gene. PCR products of EGFP gene was joined with the vector T. Weidentified the recombine-victor sequencing, which shows that the EGFP gene has been inserted into expression vector pcDNA3.0-MMx, which was named constructer combinant expression vector EGFP-Mx. Then we extracted and purified the EGFP-Mx gene. Using Electroporation transfected NIH-3T3 cells, after transfection 48h using inverted fluorescence microscope, at the same time RT-PCR to identify expression. The results showed that the constructed EGFP-Mx fusion expression vector, transfected EGFP-Mx into NIH-3T3 cells through the fluorescence inverted microscope 48 h evident Mx gene is located in the cytoplasm.It was selected by G418 after 8 days, when the NIH-3T3 cells were transfected the EGFP-Mx fusion express vector. Infected with 0.01TCID50 VSV virus to further determine the antiviral protein Mx activity and the results showed that we had constructed a right Mx gene expression vector, Mx protein had a strong anti-viral biological activity to VSV.