Cloning and expression of special F protein from human liver

Figure 1 Total RNA isolated from liver tissue of human.

Figure 2 Gel-PAGE of PCR products. Lane 1: DL-2000 marker; lane 2: PCR reactive product; lane 3: blank control.

Figure 3 Construction of the clone vector.

Figure 4 Restriction analysis of recombinant clone carrier pUCm-T. Lane1: Marker λDNA/EcoRI+ HindIII; lane 2: single enzyme digestion of recombinant plasmid pUCm-T-F/BamHI; lane 3: double enzyme digestion of recombinant plasmid pUCm-T-F/BamHI + EcoR; Lane 4: free plaszmid pUCm-T after single enzyme digestion; lane 5: RT-PCR product of total mRNA from human liver; lane 6: PCR product of recombinant plasmid pUCm-T-F; lane 7: PCR product of free plasmid pUCm-T.

Figure 5 Construction of expression vector.

Figure 6 Digestion and identification of expression vector. Pet15b-F. Lane 1: DNA Marker (DL15000); lane 2: single enzyme digestion of helper plasmid pLysS in expressed strain (pLysS/HindIII); lane 3: single enzyme digestion of expression vector pET15b-F containing the exogenous gene (pET15b-F/HindIII); lane 4: double enzyme digestion of the blank vector pET-15b-pET-15b/BamHI + NdeI); lane 5: double enzyme digestion of the expression vector pET15b-F containing the exogenous gene (pET15b-F/BamHI + NdeI; lane 6: double enzyme digestion of cloning vector pUCm-T-F containing the exogenous gene (pUCm-T/BamHI + NdeI; lane 7: double enzyme digestion of helper plasmid pLysS in the expressed strain (pLysS/BamHI + NdeI).

Figure 7 SDS-PAGE of pET15b-F under experimental conditions. Lane 1: low molecular weigh protein marker; lane 2: all strains of proteins after inducing the expression vector pET15b-F containing the exogenous gene by IPTG; lane 3: inclusion body precipitation by cleavage after inducing the expression strain containing the exogenous gene; lane 4: supernatant by centrifugation after inducing the expression strain containing the exogenous gene; lane 5: all proteins from strains not induced by IPTG containing the exogenous gene; lane 6: expression of proteins induced by IPTG containing no exogenous F protein gene; lane 7: expression of proteins induced by IPTG in a blank strain containing no expression vector.

Figure 8 SDS-PAGE of target protein in purity process. Lane 1: protein induced from bacteria; lane 2: inclusion body precipitation of all bacteria after cleavage and centrifugation; lane 3: dissolved inclusion granule after preliminary purification; lane 4: rough protein eluted by affinity chromatography; lane 5: chromatography effluent before the target protein was eluted; lane 6: first pipe of aim effluent; lane 7: second pipe of aim effluent; lane 8: last pipe of aim effluent; lane 9: blank control (the blank strain containing no expression vector).

Figure 9 Electrophoresis of target protein in all expressed and purified strains. Lane 1: low molecular weight marker; lane 2: whole bacteria induced protein; lane 3: inclusion body precipitation after cleavage and centrifugation; lane 4: supernate from all strains cleaved and centrifuged; lane 5: whole bacterial protein not induced; lane 6: expression strain containing induced blank vector (pET15b); lane 7: induced expression of proteins not containing the expression vector; l8: electrophoresis of the target protein taken out from the gels.

Figure 10 Western-blot of purified human liver F protein. Lane 1: low molecular weight marker; lane 2: precipitation of inclusion granule cleavage; lane 3: inclusion granule dissolved by carbamide; lane 4: supernatant of expression strain after cleavage and centrifugation; lane 5: expression of strain not induced; lane 6: protein of induced expression strain containing the blank vector (pET-15b, not containing exogenous gene F); lane 7: whole proteins of induced blank expression strain not containing the expression vector; lane 8: protein purified by a His-tag column; lane 9: Western-blot result of purified human liver F protein.