[Prokaryotic expression and immunogenicity analysis of phosphoglycerate kinase 1 of Candida albicans].
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Abstract:
To construct prokaryotic expression plasmids of Candida albicans gene phosphoglycerate kinase 1 (pgk-1) and examine the immunogenicity of the recombinant protein.The full-length coding sequence of pgk-1 was amplified by PCR and cloned into the prokaryotic expression vector pET30a. The 6×His-tagged protein was induced by IPTG in E.coli BL-21(DE3) and the recombinant protein was identified by SDS-PAGE and Western blotting. The BALB/c mice were immunized with the purified recombinant protein to evaluate the antigenicity of the recombinant protein by ELISA.The full-length pgk-1 gene was cloned from SC5314 genome and pET-30a-pgk-1 was successfully constructed. The recombinant protein PGK-1 was highly expressed in E.coli with a relative molecule mass of 54 810. ELISA indicated that the titer of the antibody was about 1:1024.PGK-1 was successfully expressed by prokaryotic expression system and the recombinant protein showed favorable immunogenicity in mice.Keywords:
Phosphoglycerate kinase
Antigenicity
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Objective:To express and characterize human tumor protein D52(hTPD52) protein using the proper prokaryotic expression vector in E.coli.Methods:The hTPD52 gene was amplified by RT-PCR from MCF-7 cell line,and cloned into vector pMD18-T.After sequenced,the correct fragment of hTPD52 was inserted into three different prokaryotic expression vectors,which were then transformed into E.coli.BL21(DE3),respectively.The optimal expression condition was selected.After induced with IPTG,the recombinant protein was purified with His Trap affinity chromatography,and characterized by Dot blot and Western blot.Results:The hTPD52 with isoform 3 was amplified and sequenced correctly,and the recombinant expression plasmid was constructed successfully.SDS-PAGE analysis showed that the molecular weight of hTPD52-Trx fusion protein was about 44ku.The purified protein could be used as antigen to detect the specific anti-hTPD52 antibody by Dot blot and Western blot.Conclusion:The recombinant fusion protein of hTPD52 could be expressed with high performance in E.coli,and the antigenicity of hTPD52 protein is retained for the research in the future.
Antigenicity
Myc-tag
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Objective To construct prokaryotic expression plasmids of Candida albicans gene phosphoglycerate kinase 1( pgk-1) and examine the immunogenicity of the recombinant protein. Methods The full-length coding sequence of pgk-1was amplified by PCR and cloned into the prokaryotic expression vector pET30a. The 6 × His-tagged protein was induced by IPTG in E. coli BL-21( DE3) and the recombinant protein was identified by SDS-PAGE and Western blotting. The BALB /c mice were immunized with the purified recombinant protein to evaluate the antigenicity of the recombinant protein by ELISA.Results The full-length pgk-1 gene was cloned from SC5314 genome and pET-30a-pgk-1 was successfully constructed. The recombinant protein PGK-1 was highly expressed in E. coli with a relative molecule mass of 54 810. ELISA indicated that the titer of the antibody was about 1∶1024. Conclusion PGK-1 was successfully expressed by prokaryotic expression system and the recombinant protein showed favorable immunogenicity in mice.
Phosphoglycerate kinase
Antigenicity
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Objective:To study the expression of fusion protein of HPV16capsid protein L1and transforming protein E7in E.coli.Methods :HPV16L1E7DNA fragment was amplified by poly-merase chain reaction from recombinant pUC19L1E7,the fragment was then cloned into the pMD18-T.After being digested by BglⅡ,the HPV16L1E7was inserted into the expression vector pQE30,and was transformed into E.coli M15.The recombinant vector was induced by IPTG to express the fusion protein.The antigenicity of HPV16L1E7and the expression level were detected by Western blot.Re -sults:The expression protein could reactivate the anti-L1linear epitope antibody.Conclusion:The HPV16L1E7fusion gene can express the L1E7fusion protein in E.coli sucessfully and at high level,the L1E7takes up above25%of total cell protein.
Antigenicity
Cloning (programming)
Protein A/G
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In order to express Cryptosporidium parvum calmodulin-like protein(CML)gene in E.coli BL21(DE3)and analyze the antigenicity of the recombinant protein,CML gene was amplified by PCR with cDNA of C.parvumoocysts.The amplified CML gene was cloned into pMD18-T vector and the DNA of recombinant pMD-CML plasmid was extracted.The plasmid was digested with double enzymes and the objective fragments were connected with pGEX-6p-1which had been digested with same enzymes.After identifying by double restrict enzyme digestion and gene sequence analysis,the recombinant plasmids were transformed to E.coli BL21(DE3)cells and the transformed bacteria was induced to express with IPTG.Recombinant proteins were purified by High-Affinity GST·Bind Resin affinity chromatography.Antigenicity of the recombinant proteins was analyzed by Western blot.The results showed that the prokaryotic expression vector pGEX-CML was constructed successfully and an approximate 51 ku recombinant protein rCML was expressed successfully after inducing with IPTG.The purified recombinant protein could be recognized specifically by the sera from rabbit infected with C.cuniculus.
Antigenicity
Cloning (programming)
Myc-tag
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Objective: To clone, construct and express truncated pET41a-Em18. 1 recombinant plasmid and to study its primary antigenicity. Methods: The primers of truncated Em18 were designed by DNAman biosoftware and the truncated fragment was amplified by PCR from pMD18-T/Em18, and was cloned into prokaryotic expression plasmid pET41a to construct the pET41a-Em18-1. The recombinant plasmid was analyzed by sequenceing. The rEm18.1-GST fusion protein was expressed by induction with IPTG and purified using His-tag column, and then was detected by SDS-PAGE and Western Blot. Results: The pET41a-Em18-1 positive clone was the exact recombinant plasmid and the expressed rEm18.1-GST recombinant protein can be detected as a band of 41KDa by SDS-PAGE and Western blot which confirmed that the recombinant protein could specifically react with the serum samples from patients with alveolar echino-coccosis (AE). Conclusion:The truncated pET41a-Em18.1 was constructed successfully and the rEm18.1-GST recombinant protein is expressed and shows a good antigenicity. This work has potential for use in the research of epitope analysis of Eml8 antigen.
Antigenicity
clone (Java method)
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To construct prokaryotic expression plasmids of Candida albicans gene phosphoglycerate kinase 1 (pgk-1) and examine the immunogenicity of the recombinant protein.The full-length coding sequence of pgk-1 was amplified by PCR and cloned into the prokaryotic expression vector pET30a. The 6×His-tagged protein was induced by IPTG in E.coli BL-21(DE3) and the recombinant protein was identified by SDS-PAGE and Western blotting. The BALB/c mice were immunized with the purified recombinant protein to evaluate the antigenicity of the recombinant protein by ELISA.The full-length pgk-1 gene was cloned from SC5314 genome and pET-30a-pgk-1 was successfully constructed. The recombinant protein PGK-1 was highly expressed in E.coli with a relative molecule mass of 54 810. ELISA indicated that the titer of the antibody was about 1:1024.PGK-1 was successfully expressed by prokaryotic expression system and the recombinant protein showed favorable immunogenicity in mice.
Phosphoglycerate kinase
Antigenicity
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Three overlapping fragments A,B and C of p80 gene of bovine viral diarrhea virus(BVDV) were amplified by PCR.PCR products were cloned into the expression vector pGEX-6P1 and the recombinant plasmids pGEX-6P1-p80A,pGEX-6P1-p80B and pGEX-6P1-p80C were transformed into E.coli Rosetta and BL21 cells.The target proteins of 60ku,47ku and 51ku were produced by induction using IPTG at 1.0(mmol/L).Only the p80B protein(289-477aa) reacted with BVDV positive serum in Western-blotting,indicating that an immunodominant region of the p80 protein was defined by the p80B protein.The p80B recombinant fusion protein can be used as the specific diagnosis antigen for ELISA assay.
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Objective To express 4Aβ1-15 in E.coli and purify the expressed product.Methods The 1-2 Aβ1-15 gene was amplified by PCR using recombinant plasmid pcDNA3.1-4Aβ1-15 as a template,and cloned into prokaryotic expression vector pQE30a.The constructed recombinant plasmid pQE30a-2Aβ1-15 was used as a template for amplification of 2-2Aβ1-15 gene which was cloned into plasmid pQE-30a-2Aβ1-15.The constructed recombinant plasmid pQE30a-4Aβ1-15 was transformed to E.coli M15 for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot,then purified by Ni2+-NTA affinity chromatography.Results Restriction analysis and sequencing proved that recombinant plasmid pQE30a-4Aβ1-15 was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 18 000,mainly existed in soluble form,contained about 40% of total protein in supernatant and showed specific binding to mouse anti-human Aβ40 monoclonal antibody,in which foreign proteins were basically removed after purification.Conclusion Recombinant prokaryotic expression vector pQE30a-4Aβ1-15 was constructed successfully,and His6-4Aβ1-15 fusion protein was expressed and purified.
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To construct a prokaryotic expression plasmid for extracellular domain of mouse CD40 (mCD40), express the mCD40/GST recombinant protein in E.coli, purify the mCD40/GST recombinant protein and characterize its antigenicity.Extracellular domain of mouse CD40 was amplified by PCR from cell line DC2.4 and then was cloned into prokaryotic expression vector pGEX-6P-1 to construct the recombinant expression vector pGEX-6P-1-mCD40. The expression vector was transformed into E.coli BL21 (DE3) and the fusion protein mCD40/GST was induced by IPTG. The fusion protein was purified through sepharose 4B. Then antigenicity of the purified mCD40/GST protein was verified by Western blotting and ELISA.The PCR product was verified by DNA sequencing to be consistent with the sequence of mouse CD40 on GenBank. The recombinant plasmid was identified by double digestion successfully. SDS-PAGE analysis showed the relative molecular mass of the fusion protein induced by IPTG was 45 000. Western blotting and ELISA demonstrated that the purified mCD40/GST protein had a good antigenicity.The prokaryotic expression plasmid pGEX-6P-1-mCD40 was constructed successfully. In E.coli BL21 (DE3) transformed with the plasmid, the mCD40/GST fusion protein was expressed by IPTG induction. The purified mCD40/GST fusion protein had a high antigenicity, which provides a strong support for the future study of CD40.
Antigenicity
Shuttle vector
Cloning (programming)
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Two truncated VP2 genes were amplified by PCR and were cloned into a prokaryotic expression vector pET-32a(+),and then expressed in E.coli BL21(DE3) after induced by 1.0 mmol/L IPTG.The recombinant proteins were purified and analyzed by SDS-PAGE and identified by Western blotting.The molecular weights of the recombinant proteins were about 30 000 and 33 000.The recombinant proteins were soluble proteins,its expression level was higher than VP2 protein.The result of Western blotting indicated that the recombinant proteins have special immunogenicity.Two truncated VP2 genes of canine parvovirus were expressed in prokaryotic expression system to obtain recombinant proteins which could be used to develop subunit vaccine and diagnostic reagent.
Antigenicity
Porcine parvovirus
Myc-tag
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