Cloning And Expression Of Zmp1 And Its Effects On Macrophage

Objective:To construct a prokaryotic expression plasmid for zinc-dependent metalloportease-1 (zmp1) gene from Mycobacterium and express in E.coli BL21(DE3), and purify the Zmpl recombinant protein. To construct a prokaryotic expression plasmid carrying enhanced green fluorescent protein (EGFP) gene and Mycobacterium zmp1 gene, and to express it in RAW264.7 cells. To investigate the effect of Zmpl protein on proliferation, cycle, apoptosis and phagosome-lysosome fusion of RAW264.7 cells.Methods:1. Zmpl gene was amplified by PCR from the genome of Bacillus Calmette-Guerin (BCG) as a template. The prokaryotic expression vector pET-32a(+)-zmp1 was constructed by insterting zmp1 gene fragment into multiple cloning site of pET-32a(+). The constructed pET-32a(+)-zmp1 was transformed into E.coli BL21(DE3). The recombinant Zmpl protein was expressed via IPTG induction, purified and detected by SDS-PAGE and Western blotting.2. Zmpl gene was amplified by PCR from the genome of BCG as a template. The eukaryotic expression vector pEGFP-N1-zmpl was constructed by inserting zmpl gene fragment into multiple cloning site of pEGFP-N1. The constructed pEGFP-N1-zmpl was transfected into RAW264.7 cells. Finally, the fusion protein expression of green fluorescence protein and Zmp1 was observed by fluorescence microscopy. The expression of zmpl mRNA was detected by Real-time Quantitative PCR(qRT-PCR).3. RAW264.7 cells were treated with Zmp1 recombinant protein. After 24 h treatment, the proliferation activity of RAW264.7 cells was assessed by CCK-8 method. After 48 h treatment, the cycle and apoptosis of RAW264.7 cells were assessed by flow cytometry.4. RAW264.7 cells were transiently transfected with experimental vector pEGFP-N1-zmp1. After 24 h transfection, the proliferation activity of RAW264.7 cells was assessed by CCK-8 method. After 48 h transfection, the cycle and apoptosis of RAW264.7 cells were assessed by flow cytometry.5. During the attenuated Salmonella typhimurium SL7207 strain infecting RAW264.7 cells, the effect for Zmp1 to affect phagosome-lysosome fusion was evaluated by Anti-Salmonella typhimurium/AF350 and Anti-LAMP1/Cy5 staining and fluorescence observation.Results:1. Zmp1 gene was sucessfully amplified by PCR from the genome of BCG. The recombinant plasmid pET-32a(+)-zmp1 carrying zmp1 gene was sucessfully constructed. The size and sequence of zmp1 gene were correctly by restriction analysis and sequencing. Induced with IPTG, Zmp1 recombiant protein may be expressed in E.coli. Its relative molecular mass was about 94 kDa which was the same with that of presumed fusion protein. As Western blotting showed, recombinant Zmp1 protein showed specific binding to monoclonal antibody with His tag.2. Restriction analysis and sequencing proved that the recombinant vector pEGFP-N1-zmp1 was successfully constructed. Green fluorescence was detected in transfected RAW264.7 cells using fluorescence microscope, which showed Zmp1 correctly expressed in RAW264.7 cells. As qRT-PCR showed, the expression level of zmp1 mRNA was up-regulated.3. RAW264.7 cells were treated with Zmp1 recombinant protein, the proliferation activity was lower on 24～96 hours, the cycle was mainly arrested at S phase, and the apoptosis rate was increased at early and late apoptosis rate.4. RAW264.7 cells were transiently transfected with experimental vector pEGFP-N1-zwp1, the proliferation activity was lower on 48～96 hours, the cycle was mainly arrested at S and G2 phase, and the apoptosis rate was increased at early apoptosis rate.5. During Salmonella infected RAW264.7 cell, fluorescence fusion were no differences in both Zmpl recombinant protein group and recombinant vector pEGFP-N1-zmp1 group.Conclusions:1. The prokaryotic expression vector for zmp1 gene was successfully constructed and the Zmp1 fusion protein was effectively expressed in E.coli BL21 (DE3).2. The eukaryotic expression vector for zmp1 gene was successfully constructed and the Zmp1 fusion protein was effectively expressed in RAW264.7 cells.3. Zmpl protein inhibited the proliferation, cycle and apoptosis of RAW264.7 cells, while Zmp1 protein showed no significantly blocked phagosome-lysosome fusion, which provided an experimental basis for deletion zmp1 gene to develope novel recombinant BCG vaccine.