Cloning And Expression Analysis Of Mx Gene In The Chicken

The Mx protein is a 75 KDa antiviral protein induced by IFN and some viruses. One form of the avian Mx protein was discovered to have antiviral activity (avian influenza and vesicular stomatitis). Because birds are natural hosts of avian influenza, the study of chicken Mx protein and its encoding genes are of significance. In the present study, Beijing-you (BJY), a Chinese local breed and White leghorns (WLH), a standard egg-type breed were selected. GED analysis between BJY and WLH using PCR-SSCP and PCR-RFLP, the full-length Mx cDNA of chicken was obtained and the genome structure of Mx was also analyzed; Then the differencial expression of Mx under different inducement between BJY and WLH was studied by semi-quantitative method and real time PCR.; Through optimizing the rare codon and mRNA secondary structure of translation initiation region, Mx protein was expressed in E.coli.The GTPase effector domain (GED) and, specifically, a single amino acid (asn631) plays a key role in disease resistance because a SNP resulting in an Mx protein with ser631 lacks antiviral activity. In the present study, variability at this locus was examined in two chicken breeds of distinct genetic backgrounds: Beijing-You (BJY),a Chinese local breed, and WhiteLeghorns (WLH),a standard egg-type breed.Analyses of the GED region in BJY (n=150)and WLH(n=150), using PCR-SSCP and PCR-RFLP, using a 2216 bp amplimer that includes the nucleotide responsible for the asn631 to ser631 change-of-function mutation, found significant differences in allelic and genotypic frequencies.The frequency of the A allele was significantly higher in WLH (0.8471) than in BJY (0.1613), while frequency of the B allele was 0.8387 in BJY and 0.1529 in WLH. The AA genome, encoding asn631 and responsible for antiviral activity, was significantly (P<0.01) more in WLH than in BJY populations. The gene frequencies and genotypes, based upon PCR-SSCP, were in Hardy-Weinberg equilibrium .The full length cDNA (including a key part of the promoter region, the 5'UTR, CDS and 3'UTR), corresponding to the avian Mx gene was amplified in WLH induced by polyI:C Nucleotide sequence analysis indicated that the predicted amino acid sequence of the protein included asn631. This cDNA is likely to be of value for avian transgenic research.All 14 exons of Mx were separately amplified from gDNA obtained from BJY and WLH chickens and these were consistent with the available cDNA sequences, including the 13 coding exons (48bp, 329bp, 193bp, 138bp, 155bp, 139bp, 199bp, 79bp, 123bp, 42bp, 59bp, 77bp, 243bp, 463bp). This results will provide the basis for a thorough analysisof polymorphisms of the Mx gene.In this study, a semi-quantitative method was used to detect Mx protein expression, induced by polyI:C, in chicken embryo fibroblasts from and BJY chickens. Expression of Mx protein in WLH fibroblasts varied with different concentrations of polyI:C, but no expression was observed in BJY-derived fibroblasts. Further study of expression of Mx gene in WLH, using real-time PCR, gave the same results as did the semi-quantitative method, but the precise underlying mechanism still needs additional study. Four bioengineered, bacterial expression systems were developed in order to study rare codons and mRNA secondary structure of the translation initiation region. By optimizing the rare codon and mRNA secondary structure, the Mx protein was expressed in pETRTMx, and pGEXRTMx, with detection of the specified 75KDa product. The results showed that Rosetta (DE3) strain, which can express some rare codons used in our experiment can allow Mx protein expression, and mRNA structure of lower energy improves the level of expression. In this manner, the expression of the full ORF of the gene for the Mx protein was obtained for the first time. It was apparent that appropriate expression vectors and strains were very important in designing an Escherichia coli expression system for Mx.