ABSTRACT
Low purification efficiency and incomplete characterization of male goat (buck) spermadhesins (Bdhs) prompted us to develop an effective system to produce recombinant Bdhs (rBdhs). Bdh-2 cDNA was inserted into a prokaryotic expression plasmid, pTrcHis TOPO. The pTrcHis-Bdh-2 system was constructed to produce a His(6) fusion protein in Escherichia coli Top10 cells. Recombinant clones were selected by growth in ampicillin-enriched medium, PCR amplification and nucleotide sequencing. The inserted cDNA was completely identified and recombinant protein synthesis was monitored by SDS-PAGE, followed by immunoblotting with monoclonal anti-His antibody. Expression of insoluble rBdh-2 was achieved at 0.1 to 2.0 mM IPTG, after 2 to 6 h of induction. Significantly increased production of rBdh-2 (P < 0.01) occurred with 1.5 mM IPTG after 2 h of induction, and with 0.3 mM IPTG after 4 h in culture. Among the induction times investigated, a period of 6 h gave the lowest levels of rBdh-2 production; with a 6-h incubation, there were no significant differences in rBdh-2 production for the various concentrations of IPTG tested (P > 0.05). The apparent molecular weight of rBdh-2 was 15.85 +/- 0.09 kDa, calculated by image analysis of membranes. This is similar to the theoretical molecular weight of 15.5 kDa predicted from the nucleotide sequence. Prior to this study, expression of recombinant goat spermadhesin had never been reported. Thus, an effective prokaryotic rBdh-2 expression system was developed in order to provide an adequate tool for studying biofunctions of goat spermadhesins.
Subject(s)
Gene Expression Profiling , Seminal Plasma Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/metabolism , Escherichia coli/metabolism , Genetic Techniques , Goats , Isopropyl Thiogalactoside/chemistry , Male , Models, Genetic , Molecular Sequence Data , Recombinant Proteins/chemistry , Time FactorsABSTRACT
PAL is a glucose/mannose-specific lectin isolated from Pisum arvense seeds. Previously, we demonstrated the capacity of other leguminous lectins to induce oedema formation and neutrophil stimulation. To investigate the potential pro-inflammatory activity of PAL, we have studied its ability to induce neutrophil migration into peritoneal cavities of rats and neutrophil chemotaxis in-vitro. The role of resident cells and sugar residues on PAL activity was analysed. PAL or saline (control) were administered intraperitoneally to rats, and total and differential leucocyte (macrophages, neutrophils and mast cells) counts were performed. The role of resident cells on the PAL effect was evaluated using three strategies: reducing the total resident cell population by lavage of rat cavities with saline; increasing macrophage population by treating animals with thioglycolate; and depleting mast cell population by subchronic treatment of rats with compound 48/80. PAL induced in-vitro and in-vivo neutrophil migration. In-vivo, PAL (50, 100, 200 and 300 microg) significantly (P < 0.05) and dose-dependently increased neutrophil migration by 600, 740, 900 and 940%, respectively, showing maximal effect 4 h after injection. PAL induced mononuclear cell migration. The neutrophil stimulatory effect of PAL was potentiated in animals treated with both thioglycolate and compound 48/ 80. The indirect lectin chemotactic effect was shown in rats injected with supernatant from cultured macrophages stimulated by PAL. In conclusion, PAL was shown to exhibit in-vivo and in-vitro proinflammatory activity. The in-vivo effect seemed to occur by a dual mechanism that was independent, but also dependent, on resident cells.
