Construction And Suppressive Effect Of SiRNA Expression Vector Targeting Gp37 Gene Of Avian Leukosis Virus Subgroup J In Vitro

The aim of the study was to suppress ALV-J replicating in cells by RNAi for targeting gp37 gene.Different DNA sequences targeted to gp37(encoding tegument protein TM) gene of NX0101strain 0f Avian Leukosis Virus Subgroup Jfor small interference RNAs are designed by consulting the successful exeperience of Ambion and Takara companies and utilizing web-based tool.At the same time, negative siRNA is designed according to the correspounding principles for negative design. By BLAST analysis, the selected sequences are not the same as the other ones of chick and the ones of other viruses. The sense strand of siRNA was composed of the start 19 nucleotides of the5'end of target squence. An TTCAAGAGA was added between sense strand and ansense strand , and an TTTTTT was added to the 3'end of target sequence as stop codes of the U6 promoter. Thus the target sequence was transcribed into a small hairpin RNA. Besides BamH I(GATCC) was designed at the 5'end of target sequence, Xho I ( CTCGAG) and Hind III(TTCGA) were designed at the 3'end of target sequence. After these target sequence were cloned into psilencer-2.1 U6 respectiveiy, pu6gp37-1,pu6gp37-2,pu6gp37-3, pu6 gp37-4,pu6gp37-5,pu6gp37-6, pu6gp(negative control) vectors were constructed. Then those vectors were digested with Xho I and were measured sequences individually. The results demonstrated the different vectors targeting gp37 were constructed successfully.With the help of Primer 5.0 software a pair of primers was designed accoding to the sequence of gp37 gene of Avian Leukosis Virus Subgroup J NX0101 strain published in the GenBank. And gp37(591bp) was amplified by poiy merase chain reaction (PCR) with extracted total DNA of A LV-J NX0101 strain. And the gp37gene was clond into pMD18-T Vector, pMD-gp37 was constructed. Then the pMD-gp37 was digested with Xho l and Kpn l, gp37 was cloned into pEGFP-N1, accordingly pEGFP-gp37 fusion expressin vector was constructed .Then pEGFP-gp37 plasmid DNA was transfected into secondary chicken embryonic fibroblasts(CEF)cells, the flourescence in CEF cells was observed at 24,48,72 hours respectively.The result was showed the fluorescence in CEF cells was detected at 24 hours, the most at 48 hours and gradually decreased and weakened at 72 hours. In order to detect inhibition effect, different siRNA expession vectors were respectively transfected into CEF cells with pEGFP-gp37, the fluorescence in CEF cells was observed at 24,48,72 hours. The results were showed the the fluorescence in CEF cells trested pu6gp37-2 or pu6gp37-4 trended to both decrease and weaken at 48 hours after transfection . This study provided a primary material for anti-ALV-J infection by RNAi and provided a new approach to study of the function of viral proteins in ALV-J infection.