小鼠Tnfaip8基因重组反转录病毒的构建

im: To construct recombinant retrovirus with EGFP expressing mouse tumor-necrosis-factor alpha induced protein 8(Tnfaip8) gene. Method: The expression of Tnfaip8 gene in different mouse cell lines were checked by qPCR, the one with the highest level of Tnfaip8 was chosen as cDNA source. Then full-length Tnfaip8 sequence was amplified by PCR using upstream primer containing XhoI site and downstream primer containing EcoRI site.The PCR product and vector MigR1 were ligated after double digestion. The construct MigR1-Tnfaip8 was verified by sequencing.Subsequently,the construct MigR1-Tnfaip8 was transfected into HEK293T together with packaging plasmids PCGP and VSVG using calcium phosphate transfection kit.24h and 48h later,The supernant was collected and infected HEK293T cells. The virus expression was observed under fluorence microscope and the virus titer was determined.Tnfaip8 expression in virus infected cells was examined by qPCR and Western Blot.Result: The PCR results showed NIH3T3 cells expressed the most Tnfaip8 out of all the mouse cells we checked.The electrophoresis showed the PCR fragment we got was about 650bp,which is consistent with full-length Tnfaip8 sequence.The MigR1-Tnfaip8 construct was confirmed by sequencing.The virus titre was about 5.5×108 cfu/ml。The Tnfaip8 expression in MigR1-Tnfaip8 virus infected cells was proved by qPCR and Western Blot.Conclusion: The recombinant retrovirus containing mouse Tnfaip8 gene was constructed successfully and would greatly facilitate our further study.?????