Cloning Of Chicken Mx Gene,Studying Of It's Assistance To AIV And Generation Of Transgenic Mice With Chicken Antiviral Gene Mx

Full-length sequence of Beijing white chicken (BJ)Mx gene was amplified by RT-PCR from the total RNA extracted from chick embryo fibroblast (CEF) induced by poly(I)/(C). The sequence analysis indicated that the chicken Mx gene containsed an open reading frame (ORF) of 2118 bp encoding a 705 amino acid. The amino acid at position 631, which was deduced from the amplifed Mx gene, was Asn; it was concluded from the analysis that amplified chicken Mx gene had the antiviral molecular characteristics and functional construction.The amplified chicken Mx gene was subcloned from pT-Mx to the modified vector pcDNA6.2 /EmGFP, The expression recombinant plasmid pcDNA6.2/EmGFP-Mx was constructed and then was transfected into MDCK cells. 12 hours later, the transfected MDCK cells were infected with subtype H5N1 strain, the culture supernatants were harvested at different time points after infection. The inhibitory rates of influenza virus in MDCK cells transfected with pcDNA6.2/EmGFP-Mx was 75.0% according to the HA results; The results from quantitative RT-PCR showed that the PB2 gene was reduced nearly 74.41% and 72.99% in infected MDCK cells after transfected by pcDNA6.2/EmGFP-Mx 12h and 24h, In contrast, the control plasmid did exhibit no inhibitory effect on the PB2 gene.Recombinant plasmid pcDNA6.2/EmGFP-Mx was enzyme-restricted by SmaI and NruI, a 5kb linerized DNA fragments were recovered by gel extraction, and the transgenic mice were producted by microinjection method. The transgenic mice were identified by PCR,Southern blot and detect fluorescence. Results showed that integration rate were 44.83 % and 10.34 % by PCR and Southern blot respectively. The heredity rate of Mx gene from G0 to F1 was lower 50%, so it presumed that the G0 transgenic mice were mosaic. RT-PCR analysis showed that the mRNA of GFP and Mx were expressed in all the tissues of transgenic mice, but GFP expression was very weak. The Conclusion was chicken Mx gene transgenic mice were obtained and transgenic mice strains were established by mate the transgenic mice with normal mice.Based on the plasmid pcDNA6.2/EmGFP-Mx, the EmGFP-Mx cassette was reconstructed into pLenti6/V5-DEST by BP/LR reaction, then cotransfect the 293FT cells mediated by pLenti6/EmGFP-Mx and packaging plasmids so as to produce the lentivirus. Titer the lentivirus by NIH3T3,the titer was 5.0×104TU/ml; detect the expression of Mx gene on lentiviral particles infected cells by RT-PCR, the result showed that Mx gene has transuded efficiently to NIH3T3 cells. the lentivirus could infect mammal`s cells, demonstrated that this lentiviras expression system was successfully established and it could be used to produce lentiviral particle for Mx gene delivery.