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Objective:To construct a recombinant vaccine of Schistosoma japonicum (Sj) mediated by Enterococcus faecalis (Efs, rEfs-Sj26GST vaccine), and to study the expression of Sj26GST-GST fusion protein in the recombinant vaccine. Methods:The recombinant plasmid pGEX-Sj26GST was transformed into the susceptible strain Efs ATCC47077 by electroporation to construct rEfs-Sj26GST vaccine, and the plasmid was extracted for PCR identification. After induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), the products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Results:After PCR identification, a 676 bp fragment was amplified, which was consistent with the length of Sj26GST amplification fragment. SDS-PAGE analysis showed that the relative molecular mass was 52 × 10 3, which was consistent with the band of Sj26GST-GST fusion protein. Western blot results showed that the Sj26GST-GST fusion protein expressed by rEfs-Sj26GST vaccine could be specifically recognized by the serum of Sj infected patients. Conclusion:The rEfs-Sj26GST vaccine is successfully constructed, and the Sj26GST-GST fusion protein expressed by recombinant vaccine can be specifically recognized by the serum of Sj infected patients.
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Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and is endemic in many Latin American countries. Diagnosis is based on serologic testing and the WHO recommends two or more serological tests for confirmation. Acidic ribosomal P protein of T. cruzi showed strong reactivity against positive sera of patients, and we cloned the protein after fragmenting it to enhance its antigenicity and solubility. Twelve positive sera of Chagas disease patients were reacted with the fragmented ribosomal P protein using western blot. Detection rate and density for each fragment were determined. Fragments F1R1, F1R2, and F2R1 showed 100% rate of detection, and average density scoring of 2.00, 1.67, and 2.42 from a maximum of 3.0, respectively. Therefore, the F2R1 fragment of the ribosomal P protein of T. cruzi could be a promising antigen to use in the diagnosis of Chagas disease in endemic regions with high specificity and sensitivity.
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Humanos , Western Blotting , Enfermedad de Chagas , Células Clonales , Diagnóstico , Parásitos , Sensibilidad y Especificidad , Pruebas Serológicas , Solubilidad , Trypanosoma cruziRESUMEN
Pancreatic acinar cells synthesize pancreatic lipase related protein 1 (PLRPl), which has a high degree of sequence and structural homology with pancreatic triglyceride lipase (PTL). PTL is required for efficient dietary triglyceride digestion, while the physiological role of PLRPl has not been elucidated, although some investigations have shown that its expression level is changed under some physiological or pathological conditions. Specific antigenic peptides were fused to glutathione S-transferase (GST) and purified recombinant fusion protein was used to generate polyclonal antisera by immunization of rabbits. The antisera could detect the antigen as low as 0.6 ng and PLRPI protein in 3 μg of mouse pancreatic juice extracts. The high specificity was verified in Western blot and immunohistochemistry analysis by using PLRPl knockout (KO) mice as the control. Furthermore, it was showed that food intake could increase the exocrine secretion of PLRPl into pancreatic juice. This implied that PLRPl may fulfill dietary digestion function in the digestion track.
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Nisin is a cationic antimicrobial peptide produced by some lactic acid bacteria. However, expression of nisin resistance protein (NSR) could confer nisin resistance on some non-nisin-producing Lactococcus lactis. To deeply elucidate molecular mechanism underlying NSR-mediated nisin resistance, an NSR mutant with N-terminal 38 amino acid residues deleted (NSR?38) was overexpressed in Escherichia coli by fusion with GST. Purified NSR?38 was obtained through glutathione (GSH) affinity chromatography followed by cleavage of GST tag. Putative proteolytic activity of NSR?38 was determined in vitro against nisin. Antimicrobial activity analysis revealed that nisin lost its bactericidal activity after incubation with NSR?38. Further reversed-phase high performance liquid chromatography (RP-HPLC) analysis indicated that NSR?38 displayed proteolytic activity against nisin, thus inactivating the antimicrobial peptide. The current study paves the way for in-depth functional studies on NSR.
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Objective:To obtain human S100A2-GST fusion protein for further research on human S100A2(hS100A2)protein function and its interactions with other proteins and systems.Methods:hS100A2 gene from pHAHA-hS100A2 was subcloned into an expression vector pGST-moluc to construct pGST-moluc-hS100A2.The new plasmid was identified by digestion with XhoⅠand EcoRⅠ.The recombinant BL21 was induced with IPTG to express recombinant fusion protein hS100A2-GST,which was purified by Glutathion-Sepharose 4B ball beads,identified by SDS-PAGE and Western Blot,and quantified by Bradford method.Results:After the digestion,the recombinant plasmid was cutted into two fragments,300 bp and 5kb.After treated with IPTG,the recombinant BL21 strain expressed a protein,which was about 36 kD and was recognized specifically by an-ti-hS100A2.Its yield was 5 mg/L bacterial culture.After isolated by Glutathion-Sepharose 4B ball beads,its purity was 92%.Conclusion:hS100A2-GST expression plasmid pGST-moluc-hS100A2 was constructed successfully.hS100A2-GST fusion pro-tein could be expressed in Escherichia coli with higher yield and isolated with higher purity,which lays the foundation for the follow-up of the hS100A2 research.
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C8orf32 is a gene which has not been functionally characterized,the mRNA level of this gene is significantly higher in breast cancer tissues than that in normal breast tissues.The amplified cDNA fragment was inserted into the pGEX-6P1 vector fused with the upstream GST gene.The expression vector was transformed into the E.coli BL21(DE3) strain and expression of GST-C8orf32 fusion protein was induced by IPTG..After removal of GST tag by site-specific protease,the C8orf32 protein fused with an eight amino acid peptide tag was obtained.The purity of recombinant C8orf32 protein was about 95%.The identity of the purified protein was confirmed by N-terminal sequencing and tandem mass spectrometry.The polyclonal antibody was prepared by immunizing the New Zealand white rabbits with C8orf32 protein.The polyclonal antibody was proved to recognize the C8orf32 protein correctly.The purified C8orf32 protein can be used for structural and functional studies and the polyclonal antibody can be used for tissue specific protein expression profiling.
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Mimecan belongs to a family of leucine-rich proteoglycans that are secreted into the extracellular matrix. In order to investigate the function of mimecan, the coding region of mimecan was amplifed from a human pituitary cDNA by PCR and the recombinant prokaryotic expression vector pGEX-M was constructed. The vactor was transformed into E.coli BL21(DE3)and the GST-M fusion protein of 38 Kd was ecpressed in the bacteria under induction of IPTG. After purification, the fusion protein was infucted into New Zealand rabbits to prepare polyclonal antibody. The antibody was tested by Western blotting for their specificity and sensitivity. Using the antibody it was found the mimecan was expressed highly in certain types of human pituitary tumor tissues. These results make it possible for studying the biological function of mimecan.
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Objective:To construct a recombinant prokaryotic expression vector pGEX-5X-3/hIL-17F and express it in E.coli and to explore the biological activities of human IL-17F fusion protein.Methods:The coding sequence of the mature human IL-17F(minus the signal peptide) was amplified from pUCm-T/hIL-17F by PCR and subcloned into the prokaryotic expression vector pGEX-5X-3 to express glutathione S-transferase(GST) fusion protein. The fusion protein was induced in E.coli BL21 by IPTG and purified by standard methods reported in prokaryotic system. The purified GST-hIL-17F fusion protein was identified by Western blot. The proliferation of ECV304 cells was observed by incubating them with soluble GST-hIL-17F fusion protein by MTT assay. The concentrations of IL-6, IFN-? and TNF-? in the supernatants of ECV304 cells were determined by ELISA. The effect of GST-hIL-17F on the angiogenesis of the chick chorioallantoic membrane was assessed by CAM assay.Results:A 41 kD fusion protein was effciently induced in E.coli BL21 by IPTG, accounting for about 55% of the total bacterial protein. The purified GST-hIL-17F fusion protein was identified by Western blot. GST-hIL-17F fusion protein had obvious biological activity to inhibit the proliferation of ECV304 cells and enhance IL-6 secretion. GST-hIL-17F had a marked antiangiogenic activity.Conclusion:The preliminary study of hIL-17F recombinant prokaryotic expression and its antiangiogenic effect has been successful, which lays a foundation for future research on the mechanism of antiangiogenesis and clinical application of recombinant hIL-17F protein.
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This study was carried out to obtain the 3beta-hydroxy-5-ene steroid dehydrogenase (3beta-HSD) which is contained in human trophoblasts. To produce 3beta-HSD cDNA, reverse transcription-polymerase chain reaction (RT-PCR) with the total RNA of human placenta was performed and RT-PCR products was detected at 1.2 Kb. After cloning 3beta-HSD cDNA in pGEX 4T-2 as expression vector, the recombinant DNA was transformed to E-coli and glutathione-s-transferase (GST) fusion protein was inducted with isoprophyl thiogalactoside (IPTG). Inducted GST fusion protein was overexpressed in cytoplasm of E-coli. The obtained GST fusion protein was characterized with antibody against 3beta-HSD by Western blot analysis and purified by affinity chromatography using glutathione sepharose 4B column. And then the GST fusion protein was cleaved by thrombin and The cleaved protein, 3beta-HSD, was detected at 43 KDa by SDS-PAGE.