Establishment And Identification Of MDCK Cell Lines Stably Expressing Influenza Virus HA,NA Gene

In order to establish the MDCK cell lines stably expressing influenza virus HA, NA gene, the recombinant plasmid pMX-PR8HA,pMX-GDHA,pMX-GDNA and pMX- H9N2NA was constructed respectively by inserting the ORF of PR8HA gene between restriction enzyme sites AgeI and NotI,inserting the ORF of GDHA,GDNA and H9N2NA gene between restriction enzyme sites NotI and XholI on plasmid pMX, which contained the puromycin resistance gene. Four vectores of pMX-PR8HA, pMX-GDHA, pMX-GDNA and pMX-H9N2NA were constructed . To generate retroviruse like particles, pMX-PR8HA,pMX-GDHA,pMX-GDNA and pMX-H9N2NA was co-transfected to 293T cell respectively with the plasmids expressing vesicular stomatitis virus G(VSVG), Gal/Pol and NF-B protein. The retroviruse like particles in the supernatant of transfected 293T cell culture was used to infect MDCK cell. The MDCK cell clone candidates stably expressing influenza virus HA, NA gene were selected by using the existence of puromycin. After ten passages, the HA,NA gene and protein in the cells were still detectable through IFA, PCR, RT-PCR and western blot tests. The results indicated that the MDCK cell lines stably expressing influenza virus HA, NA gene were established, which offered an opportunity to study influenza virus as a viral vector to express exogenous genes. The NA gene deletion virus particles could were saved by transfection of the pMX-GDNA with seven segments of influenza plasmid, Obtained in the proliferation of the cell lines, lack of transfer on behalf of the NA of influenza virus. By this study, the expression of influenza virus protein in stable cell lines on the proliferation of highly pathogenic influenza virus gene deletion method, So lower levels of biological safety laboratories to carry out the highly pathogenic influenza virus research foundation. In addition this study also determined to build H9N2NA cell lines NA genes of wild strains, and the strain of the NA gene sequence was analyzed.