Objective To construct lentiviral vector of p62 gene over-expression,and stably express p62 gene in human monocytic leukemia cells 1(THP-1),and to provide a way to study the role of p62 gene at the cellular lev-el.Methods The p62 gene fragment was amplified by polymerase chain reaction(PCR),and the amplified prod-uct was ligated to the linearized pcDNA3.1-Flag-PCDH10 lentiviral vector.After identifying with PCR,the PCR product was cotransfected with the packaging plasmid into human embryonic kidney cells 293(HEK 293T).THP-1 cells were infected with recombinant lentivirus.Positive cell clones were screened by ampicillin.Western blot and real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)were used to detect THP-1 cell lines with high p62 expression(overexpression group)and THP-1 cell lines transfected with empty plasmid without p62 gene(control group).The expression levels of TNF-α,IL-1β and Cxcl1 after K.p.infection were detected by RT-qPCR.Results The p62 gene fragment was successfully obtained by PCR and ligated to pcDNA3.1-Flag-PCDH10 vector.PCR confirmed that p62-pcDNA3.1-Flag-PCDH10 recombinant plasmid was constructed successfully.Am-picillin-resistant cell lines were selected after lentiviral infection of THP-1 cells.The results of Western blot analysis showed that the THP-1 cells with drug sieve survival increased the expression of P62 protein compared with the con-trol cells(P<0.001),and RT-qPCR analysis showed that the relative mRNA expression of p62 increased(P<0.001).THP-1 cells with high expression of P62 were successfully constructed.The levels of TNF-α、IL-1β and Cxcl1 from THP-1 cells with high expression of P62 significantly increased after infection with K.p.(P<0.01).Conclusion P62-pcDNA3.1-Flag-PCDH10 vector and THP-1 cells with high expression of P62 can be successful-ly constructed by three-plasmid packaging system,which provides a basis for the study of p62.
维普
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