The Mutants Construction And Expression Of Agno-1a In Avian Polyomavirus Late Genes

In order to study the translational regulation of agno-1a gene in APV-1 late polycistronic mRNA,the recombinant cDNA viruses were designed and constru cted,which were similar to the wild-type APV-1 virus in genomic structure but with the point mutation at translation initiation codon ATG of the APV-1 late gene agno-1a.Firstly,a large amount of pHL1003 plasmid DNA of APV-1 cDNA virus was extracted.Plasmid pHL1003 DNA was digested with restriction en donuclease AccI(partial enzyme digestion)and EcoRI(complete digestion)in order t o obtain a large carrier fragment of pHL1003.The small target DNA fragments contai ning an ATG point mutation(ACG or CTG)of the agno-1a initiation codon,which were introduced in designed the primers,were amplified by PCR.And they were doubledigested with restriction enzymes AccI and EcoRI,then ligated into the pUC19 vector to get the accurate small DNA fragment contained with ACG or CTG.Finally,the sm all fragment with the mutation was ligated with the large vector fragment of pHL1003 by T4 ligase to construct a recombinant cDNA virus that could be expressed in eukar yotic cells.The constructed cDNA viral plasmid contained ATG mutant was transfect ed into chicken embryo fibroblasts,and the expressed protein of the recombinant viral genes were detected by Western blot.The desired carrier fragment and the target sma ll fragment were achieved by digested plasmid pHL1003-GFP and pHL1003 mutants with the restriction enzymes NdeI and NarI.Then,the mu tation target fragment(ATG?CTG or ATG?ACG)was ligated with the pHL1003-GFP carrier fragment to construct an expression vector containing the downstream rep orter gene EGFP(i.e.the GFP gene was instead of the VPs gene).After transfecting t he GFP recombinant DNA into quail embryonic fibroblasts,the fluorescence of expres sed GFP was observed by a fluorescence microscope.The results showed that plasmidpHL1003 expressed three proteins,VP1,VP2 and Agno-1a,and VP1 was the largest amount of the proteins;plasmid pHL1003-CTG also expressed Agno-1a and VP1 proteins,but the amount of VP1 and the proportion of VP1:Agno-1a were much less than the wild-type pHL1003.After three kinds of APV-1 cDNAGFP expression plasmids were transfected into quail fibroblasts individually,the amo unt of the intracellular fluorescent protein transfected by pHL1003-GFP was much higher than that by the plasmids pHL1003-GFP-CTG or pHL1003-GFP-ACG.Therefore,the agno-1a ATG mutation in the late APV-1 gene significantly affects the downstream genes translation.This suggests that the A gno-1a protein as a leader of the viral late polycistronic genes may play a key role in the tr anslational regulation of its downstream genes(GFP or VPs).