| Objective: To investigate secreting of proinflammatory factor TNF-α, IL-1β andHMGB1and apoptosis induced by the gonococcal recombinant protein NspA inTHP-1cells.Methods: The gene sequence of the gonococcal nspA was harvested fromGenbank,with the WHO-E strain genomic DNA as template, the target fragment wasamplificated by PCR, after treatment by restriction enzyme digestion, the product wasinserted into the prokaryotic vector pGEX-6p-1and identified by sequencing, thentransformed into the expression host strain E. coli BL-21, with different concentrationIPTG (0.2、0.5、0.8、1.0、1.2mmol/L) to induce the protein to express at differenttemperatures(13℃、20℃、25℃、30℃、37℃),and the expression products wereanalyzed and identificated by SDS-PAGE and Western blot; the recombinant proteinwas purified by GST-Bind column, protein concentration was determinated by the BCAkit; polymyxin B was used to remove endotoxin in the recombinant protein. THP-1wasstimulated by NspA protein with different time besides different concentrations to assaythe secretion of inflammatory cytokines HMGB1, TNF-αand IL-1β by ELISA. THP-1cells were treated with different concentrations of NspA for different time, afterAnnexinV-EGFP-PI staining, observed apoptotic morphological changes withfluorescence microscopy, and accounted percentage of apoptosis with flow cytometry.Results: The nspA gene target fragments were amplicated from Neisseriagonorrhoeae WHO-E strains by PCR, pGEX-6p-1/nspA prokaryotic vector was builtand confirmed by restriction enzyme digestion and the sequence was exactly the same as GenBank GI:215260662. The NspA soluble protein was obtained at25℃for4.5hwith0.8mmol/L IPTG concentration in recombinant expression strain, the molecularweight is about44kDa. The supernatant of the bacterium was selected after sonicatingand purified by GST-Bind resin affinity. Protein concentration was detected by theBCA kit as463μg/mL, and the content of endotoxin removed by polymyxin B wasless than0.5EU/mL after the Limulus test. Different levels of IL-1β, TNF-α andHMGB1were induced by2~32μg/mL NspA recombinant protein present asdose-dependent manner in THP-1cells, but also changes with the increase ofstimulation time. When the NspA stimulus concentration was8μg/mL for48h, theamount of TNF-α reach to peak as138pg/mL; and there was significantly differencesWith PBS/GST control groups and the other parallel groups (P <0.05). When theNspA stimulus concentration was4μg/mL for24h,8μg/mL for48h, the amount ofIL-1β respectively reached to the peak as56pg/mL, and there was significantlydifferences With PBS/GST control groups and the other parallel groups (P<0.05).HMGB1secretion was increasing with treating time of NspA, and there wassignificantly differencs With PBS/GST control groups and the other parallel groups(P <0.05). When NspA stimulated for60h, HMGB1secretions reached to the peak.4~64μg/mL NspA stimulate THP-1cells for different time, after Annexin V-EGFP-PIfluorescent staining and observation with fluorescence microscopy, it showed thatwith extended treating time and increased concentration of protein to stimulate, theapoptosis was even more serious.16μg/mL treating for24h, the apoptotic rate wassignificantly hightein; the apoptosis was detected by flow cytometry, and resultsshowed that with the prolongation of time and increased protein concentration, therewere obvious changes about the percentage of apoptosis. when using16μg/mL NspAto treat for24h, the apoptosis percentage reaches to its peak at18.27%, and there wassignificantly different With PBS/GST control group and the other parallel group (P<0.05).Conclusion:1.The prokaryotic expression vector pGEX-6p-1/nspA was constructed, and pureNspA recombinant soluble protein was obtained. 2. NspA recombinant protein could promote secreting inflammatory cytokines IL-1β,TNF-α and HMGB1in THP-1cells.3. NspA recombinant protein could induce apoptosis in THP-1cells... |
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