| Objective: This study was dedicated to construct porB/pGEX-6p-1prokaryoticexpression vector, express and purify recombinant fusion protein and analyze thecapability that PorB induced human monocytic cell line (THP-1) to produceproinflammatory cytokines IL-β，TNF-αand HMGB1，explore the effect of PorB onapoptosis.Methods: PorB gene was obtained from Genbank and amplified by PCR with DNAof Neisseria gonorrhoeae E strain as templates, then subcloned into the prokaryoticexpression vector pGEX-6p-1to construct the recombinant plasmidporB/pGEX-6p-1,and identified by restriction enzyme digestion and sequencinganalysis. The recombinant plasmid was transfected into Ecoli competent cellBL-21(DE3) to express recombinant protein and explore the optimal expressionconditions. The recombinant protein was refolded by Urea concentration gradientrenaturation method then purified by GST Bind resin. The concentration of thepurified protein was determinated by BCA protein assay kit. PolymyxinB andEndotoxin contant limulus kit were used to remove and determinate endotoxincontamination respectively. Levels of inflammatory cytokines IL-1β,TNF-αandHMGB1were measured,which secreted by THP-1stimulated with the recombinantprotein. AnnexinV-EGFP-PI staining was used to detect the apoptosis of THP-1treated by time-dependent and dose-dependent manners.Result:1) PorB gene was amplified with Neisseria gonorrheae E stain as template and subcloned to the expression vector pGEX-6p-1.It was proved that therecombinant plasmid was successfully constructed, which identified by restrictionenzyme digestion and DNA sequencing.2) Although PorB are expressed with IPTG inducing the Ecoli-BL-21expressionbacteria under different temperature. Most was only expressed in the precipitate,there was only small part which expressed in the precipitate. After refoldingrecombinant protein with urea concentration gradient, recombinant protein waspurified by GST Bind resin. SDS-PAGE analysis showed that there was only aband, molecular weight of59KD, which consistent with the size of the targetband. Limulus test showed that the endotoxin level was less than0.05EU/ml afterremoving endotoxin with polymyxin B.3) The production of inflammatory cytokines IL-1β,TNF-αand HMGB1secretedby THP-1were determinated by ELISA,which was stimulated by recombinantprotein in time-dependent and dose dependent manners. ELISA analysis showedthat the level of inflammatory cytokines IL-1β,TNF-αand HMGB1reach apeak at （27.10±1.23）pg/ml，（109.44±3.53）pg/ml，（320.09±9.67）pg/mlrespectively by5μg/ml of PorB，which was significantly higher than othergroups (P<0.05) after treatment for24h then decreased in dose dependent manner.Production of IL-1β,TNF-αreach peak at （39.94±2.35）pg/ml，（223.14±7.27）pg/ml，which was significantly higher than other groups (P<0.05) after treatmentfor36h while the level of HMGB1have been increasing from6h to48h.4) Flow cytometry and fluorescence microscopy were used to detect apoptosis ofTHP-1which was treated in dose-dependent manners with AnnexinV-EGFPApoptosis detection kit.(PBS and GST tagged protein as negative control).Apoptosis results showed that apoptosis rate reach peak at (23.13±2.01)％by7μg/ml of PorB, which was higher than other groups (P<0.05) after treatment for24h and reach the peak at (20.75±2.03)％，which was higher than other groups(P<0.05) when treated in time-dependent manner with7μg/ml of PorB. Conclusion:1) Secreting of IL-1β,TNF-α and HMGB1could be enhanced by therecombinant protein PorB in THP-1.2) Apoptosis in THP-1could be induced by the recombinant protein PorB indose-dependent and time-dependt manners. |
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