The Construction Of Zmp1 Lentiviral Vector And Its Effect On The Autophagy Function Of RAW264.7 Cells

Objective:1.To construct Lentiviral vector LV2-zmp1 of Zmp1,and infect macrophage RAW264.7 cells with recombinant LV2-zmp1 lentivirus to make Zmp1 gene stable expression in macrophage RAW264.7.2.To induce autophagy of macrophages RAW264.7 with rapamycin or inhibit autophagy with hydroxychloroquine,and observe the effects of Zmp1on autophagy of macrophage RAW264.7 infected by recombinant Lentivirus LV2-zmp1.Methods:1.Using the M.tuberculosis genome as a template,a zinc-dependent metalloproteinase 1(Zmp1)gene was amplified by PCR and cloned into the lentiviral expression plasmid LV1 to obtain the recombinant lentiviral expression plasmid LV1-zmp1.Lentiviral expression plasmid LV1-zmp1,recombinant shuttle plasmid pGag/Pol,secondary packaging plasmid pRev,and p VSV-G four-plasmid were co-transfected into 293T cells for virus packaging.72 hours after transfection,viral fluids were collected and titered to obtain a drop.LV2-zmp1 recombinant lentivirus with a degree of 1x10~8TU/ml.RAW264.7 cells were infected with LV2-zmp1 recombinant lentivirus.After infection for 96 hours,the infection of GFP-expressing virus was observed by fluorescence microscope.Total RNA was extracted and cDNA was synthesized with reverse transcription.Zmp1 m RNA expression was detected in RAW264.7 cells by RT-PCR.The expression of the protein was extracted from RAW264.7 cells and Western blot was used to detect the expression of Zmp1 protein.2.Lentivirus LV2-zmp1 infected mice macrophage RAW264.7,while rapamycin induced RAW264.7 autophagy and hydroxychloroquine inhibited autophagy of RAW264.7,and the number of autophagosomes was observed by transmission electron microscopy.The expression of autophagy-related genes Atg5,Atg8 and Atg12 was detected by RT-PCR,and the expression of autophagy-related proteins BeclinI,LC3II and P62 was detected by Western blot.The expression differences were used to reflect Zmp1 on effect of macrophages RAW264.7 autophagy.Results:1.Successfully constructed lentiviral expression plasmid LV1-zmp1;LV2-zmp1 recombinant lentiviral vector carrying Zmp1 gene was obtained by co-transfection of four plasmids into 293T cells;RAW264.7 cells were infected with recombinant lentivirus LV2-zmp1 and green fluorescence expression was observed under fluorescence microscope.In LV2-zmp1-infected cells,high mRNA and protein expression of Zmp1 were detected by RT-PCR and Western blot.2.Compared with the control group without Zmp1 expression,the number of autophagosomes in rapamycin-induced RAW264.7 cells was significantly reduced in the Zmp1 recombinant adenovirus LV2-zmp1 group under transmission electron microscopy;The results of RT-PCR showed that the expression levels of autophagy-associated genes Atg5,Atg8,and Atg12were lower than control,and the differences were statistically significant(P<0.05).The results of Western blot showed that expressions of autophagy-related proteins BeclinI and LC3II were reduced,the expression level of P62was increased,and the differences were statistically significant(P<0.05).After autophagy inhibitor hydroxychloroquine treatment,the expressions of autophagy-related proteins BeclinI and LC3II were down-regulated,and the expression of P62 was increased by Western blot.The changes in recombinant adenovirus LV2-zmp1 group were more obvious,the differences were statistically significant(P<0.05).Conclusion:1.Successfully constructed recombinant lentiviral vector LV2-zmp1,which can infect RAW264.7 cells and stably express Zmp1 molecules.2.Mycobacterium tuberculosis Zmp1 affects the autophagy function of macrophages by inhibiting the expression of autophagy-related molecules and inhibiting the formation of autophagosomes.