Prokaryotic cloning expression and immunologic analysis of suppressor of cytokine signaling -1 gene in rats
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Objective To clone and express the suppressor of cytokine signaling-1 gene (SOCS-1)in rats. Methods PCR product of full-length coding sequence of rat SOCS- 1 was cloned into a prokaryotic expression plasmid [pET-28a(+)] to construct the recombinant expression plasmid [pET-28a(+)-ratSOCS-1].Escherichia coli BL-21/DE3 was tranformed into the recombinant plasmid and expressed with isopropyl-β-D-thiogal (IPTG) induction. Recombinant proteins in the expression products were purified by Ni-IDA affinity chromatography. SDS-PAGE was used to verify the expression products and the recombinant protein. The purified recombinant protein was used to sensitized New Zealand rabbits until the last immunization at an samples which were then determined for antibody titers and separated for sera. Total proteins of peripheral blood WBC were prepared with RBC lysis method. Negative serum (from New Zealand rabbits not immunized with recombinant protein), immunized serum and rabbit anti-histidine labeled antibody were transferred to separate bands and the immunologic activities of recombinant proteins were determined with Western blotting. Results PCR, double enzyme digestion and DNA sequencing demonstrated successful construction of pET-28a(+)-ratSOCS-1 plasmid. The results of SDS-PAGE showed formation of an obvious protein band about 26 000 in relative molecular weight by both Escherichia coli transformed with recombinant plasmid and samples of precipitated bacterial debris from ultrasonic degradation. While characteristic single band from purified recombinant proteins obtained by affinity chromatography was shown to be consistent with this band, no protein expression in supernatant of bacterial debris from ultrasonic degradation was observed at the same location. In addition, Western blotting demonstrated the competency of immunized serum as it clearly recognized the recombinant protein, the 24 000 band of lysed protein from rat peripheral blood leukocytes, and rabbit anti-histidine labeled antibody, generating three distinct bands with relative molecule weights of 26 000, 24 000 and 26 000 respectively. Conclusions Rat SOCS- 1 can be successfully expressed in Escherichia coli (BL-21/DE3). The purified recombinant protein of SOCS-1 may have strong immunogenicity and immunocompetence.
Key words:
Suppressors of cytokine signaling- 1; Plasmids; Gene expression; Chromatography,affinity; ImmunocompetenceKeywords:
Myc-tag
Chicken IL-18 mature protein gene was amplified from IL-18 recombinant plasmid by PCR,and was subcloned into prokaryote expression vector pPROEX~(TM)HTa and then was transformed into E.coli DH5α and was induced with IPTG at 37 ℃,SDS-PAGE analysis showed induced products about 26 ku.The recombinant mChIL-18 was expressed in form of inclusion body with the yield accounting for(21.95%) of total bacterial proteins.The matural protein of Chicken IL-18 was purified by Ni-NTA resin.The antiserum was obtained by immunizing SPF chicken with the purified recombinant protein.Western blot result showed the antiserum raised against the recombinant mChIL-18 in chicken could react to protein expressed specifically.ELISA detection showed the antigenicity of the fusion protein was satisfactory.
Antigenicity
Inclusion bodies
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Objective To clone mouse sperm Sp17 gene and fulfill its expression in E. coli BL21(DE3). Methods The coding sequence of mouse protein Sp17 was amplified from mouse testis RNA through reverse transcription-polymerase chain reaction (RT-PCR), and subsequently cloned into pET-28a (+). Recombinant Sp17 was then expressed in E. coli BL21 (DE3) with induction of IPTG and analyzed by SDS-PAGE and Western blotting. The mice were immunized intranasally with the recombinant proteins and the titers of specific antibodies IgG in serum were detected. Results Sequencing and restriction digestion of the recombinant plasmid demonstrated that the coding sequence of mouse protein Sp17 was successfully cloned. SDS-PAGE and Western blot analysis showed that a recombinant protein with molecular weight of 24 000, which is corresponding to the theoretical molecular weight of Sp17 protein, existed in the extract of E. coli BL21 (DE3) cells. After immunization, Sp17-specific antibody IgG response was present in the mice. Conclusion The coding sequence of mouse Sp17 was cloned into pET-28a (+) plasmid vector, and high level of recombinant Sp17 protein expressed in E. coli BL21 (DE3) was obtained. The recombinant Sp17 protein can induce Sp17-specific antibody IgG response.
Myc-tag
Coding region
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【Objective】 A polyclonal antibody of lysostaphin(lys) protein was prepared to lay a foundation so that the expression of the lys gene could be detected in transgenic cells,embryos and transgenic animals.【Method】 The mature peptide of lys gene was amplified by PCR from the PEPB plasmid with lys gene sequence inside and then cloned into a prokaryotic expression vector pET-28a to construct a recombinant plasmid pET28a-Ly.The pET28a-Ly plasmid was transformed into E.coli BL21(DE3) cells to produce the recombinant lys protein with His tag by inducing 1 mmol/L IPTG for 6 hours.The recombinant lys protein was purified by using a His-tag fusion protein purification kit and injected into a few rabbits to get the specific polyclonal antibody.The specificity and antibody titer of the polyclonal antibody was confirmed by western blot and ELISA.【Result】 The recombinant lys protein was efficiently expressed in E.coli BL21(DE3) after induction with IPTG.3.05 mg/mL of the protein concentration was obtained after purification.Antibody titer of 1∶27 000 was detected by ELISA.The results of Western blot showed that the antibody specificity was better.【Conclusion】 The prokaryotic expression vector pET28a-Ly was constructed successfully.The prepared polyclonal antibody of lys protein has a high titer and good specificity.
Polyclonal antibodies
Lysostaphin
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Objective To construct and express the fusion gene of sperm protein 17(Sp17)and IL-5,and to analyzed the immunological characteristics of the fusion protein.Methods The IL-5 gene,amplified from the plasmid pGEM-1-IL-5 by PCR,was cloned into the plasmid pET/Sp17 and the fusion gene Sp17-IL-5 was constructed.Then Sp17-IL-5 recombinant protein was express in E.coli BL21(DE3)and analyzed by SDS-PAGE and Western blotting.Antibodies against Sp17 were detected in the sera of mice immunized with recombinant protein Sp17-IL-5,Sp17,or PBS intranasally.Results Sequencing and restriction digestion of the recombinant plasmid demonstrated that the plasmid pET/Sp17-IL-5 was successfully constructed.SDS-PAGE and Western blotting showed that the recombinant fusion protein had a molecular weight of 39 KD;the purity of recombinant protein Sp17-IL-5 was 91% after being purified using Ni2+-ANT affinity column;and after immunization,Sp17-specific antibody IgG response was present in the mice.Conclusion The coding sequence of Sp17-IL-5 is cloned into pET-28a(+)plasmid vector,and high level of recombinant Sp17-IL-5 protein expressed in Escherichia coli BL21(DE3)is obtained,which can induce Sp17-specific antibody IgG response.
Myc-tag
FLAG-tag
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To express and purify the recombinant nucleocapsid protein(N-protein) of SARS coronavirus and to analyze its induction of immune responses induced by this protein,the right gene fragment encoding the nucleocapsid protein in the recombinant clone pMD18-N was digested with restrictive endonucleases and was subcloned into prokaryotic expression vector pET-2a(+).The recombinant plasmid pET-N was then transformed into E.coli BL21,and the recombinant clone was characterized by PCR,and digested with restriction endonucleases;meanwhile the positive clone was induced with IPTG to express the target protein,this protein was characterized by SDS-PAGE;the recombinant protein was purified from E.coli cell lysate by metal-chelating chromatography,and the immunological activities of this protein were analyzed by immunoblotting and the immune responses induced in mice.It was demonstrated that the pET-N was constructed through subcloning the right insert of N-protein into pET-23a(+),and the recombinant protein could be expressed in clone containing pET-N when induced with IPTG and the expressed protein was purified with metal-chelating chromatograpjhy.The purified protein reacted with sera of mice immunized GST-N and could induce a moderate degree of immune responses in mice.It is concluded that the prokaryotic expression plasmid pET-N has been successfully constructed,and the purified recombinant protein shows good antigenicity.
Subcloning
Myc-tag
clone (Java method)
FLAG-tag
Antigenicity
Protein A/G
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To further understand the properties of the 32 kDa HIV-1 integrase(IN)required for catalyzing the insertion of viral DNA into host cell genome,the expression system that could express the HIV-1 integrase 142-288 site amino acid protein in prokaryotic expression vector efficiently was constructed,and the antigenic function of this protein was analyzed. The HIV-1 IN cDNA fragment encoding amino acid 142-288 site was amplified by PCR. The PCR product was digested with BamHI and Xhol and cloned into vector pGEX-4T1,and the recombinant plasmid pGEX-4T1-IN-142-288 was confirmed by double enzyme digestion and sequencing,transformed to E. coli BL21(DE3)and then the bacterial cultures were induced with IPTG.Recombinant protein expressed was purified through a glutathione-Sapharose 4B column and the purified IN 142-288 protein was analyzed with SDS-PAGE and Western blotting. Three BALB/c mice were immunized with the purified IN-142-288 protein,and after 3 boostings,the antisera obtained from the immunized mice were tested with ELISA and Western blotting using the purified His-tagged IN(wild type)as antigen. It was demonstrated that the length of the inserted gene fragment in plasmid pGEX-4T1-IN-142-288 was 438 bps after double enzyme digestion,and a 40 kDa protein band could be found in SDS-PAGE and Western blotting. As revealed from ELISA assay,high titered antibodies against the recombinant protein in these 3 immunized mice could be demonstrated and the antibody produced by one of the immunized mice could recognize the denatured wild type HIV-IN transferred to the NC membrane in Western blotting.It is apparent that the recombinant prokaryotic expression vector pGEX-4T1-IN 142-288 was successively constructed and the recombinant protein expressed from E. coli BL21 was proved to be excellently antigenic.
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Objective To explore the cloning,prokaryotic express and purification of the receptor for activated C kinase 1(RACK1) gene of RH strain of Toxoplasma gondii,and to analyze the antigenicity of recombinant protein.Methods Total RNA was extracted from tachyzoites of RH strain of T.gondii.The open reading frame of TgRACK1 gene was amplified with a pair of specific primers which was designed according to the coding sequence of TgRACK1 gene(GenBank accession No: AY547291),the product of RT-PCR was digested with double restrict enzyme and ligated into a pGEX-6p-1 vector.The recombinant pGEX-6p-1-TgRACK1 plasmid was transferred into E.coli DH5α and the positive clones were selected through the colony-PCR and confirmed by the double restrict enzyme digestion and sequencing.The successful pGEX-6p-1-TgRACK1 construct was transformed into E.coli BL21(DE3) and induced with IPTG to express.The products of expression were analyzed through SDS-PAGE followed by Coomassie blue staining and the recombinant TgRACK1 was purified by using glutathione-triethyleneglycocyl-sepharose 6B.Western blotting assay with GST primary antibody and rabbit anti-T.gondii serum were used to confirm the expression of rTgRACK1 and analyze its antigenic properties.Results The product of RTPCR was 970 bp.The recombinant plasmid pGEX-6p-1-TgRACK1 was confirmed successfully by colony-PCR,double restrict enzyme digestion and sequencing.A recombinant protein with a rough molecular weight of 63 kDa was analyzed via SDS-PAGE followed by Coomassie blue staining.The GST tag in rTgRACK1 and the antigenicity of rTgRACK1 were detected efficiently by Western blotting analysis with the GST primary antibody and with the prepared antiserum against TgRACK1 respectively.The isolated protein was water soluble and showed 95% purity based on SDS-PAGE and gel imaging analysis.Conclusion The TgRACK1 gene is cloned,the recombinant protein TgRACK1 is produced in E.coli and purified and maintained the specific antigenicity.
Antigenicity
Cloning (programming)
Coomassie Brilliant Blue
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Objective
To investigate the prokaryotic expression and immunoreactivity of BspE, a type Ⅳ secretion protein of Brucella, and the effect of recombinant protein BspE on cytokines.
Methods
According to the BspE gene of Brucella M5-90 published in GenBank, the gene fragments were synthesized by a company and then ligated into PUC57 vector for sequencing. The sequenced gene was cloned into a prokaryotic expression vector pET-28α and transformed. Induced expression was performed in E. coli DE3 competent cells. The obtained target protein was purified by a Ni-NTA affinity column, and its reactogenicity was analyzed by Western blotting. Mouse RAW264.7 cells were treated with 25 g/L BspE recombinant protein for 12, 24, 48 h, and the control group was treated with the same amount of BSA instead of BspE, and enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin (IL)-1β level.
Results
The recombinant expresed plasmid of pET-28α-BspE was successfully obtained. The results of Western blotting showed a single band with a relative molecular mass of about 30.1 × 103, and the recombinant protein BspE had good reactogenicity, and IL-1β levels (ng/L) were significantly elevated by the recombinant protein BspE (12 h: 43.27 ± 2.13 vs 30.24 ± 1.66, 24 h: 57.78 ± 3.44 vs 41.22 ± 1.22, 48 h: 72.52 ± 3.04 vs 46.77 ± 2.75, t= 8.38, 7.86, 10.89, P < 0.05).
Conclusions
BspE recombinant protein has better immunoreactivity and can increase the expression level of IL-1β in mouse macrophages. This study provides a scientific basis for the role of effector proteins in the pathogenesis of Brucella.
Key words:
Brucella; BspE gene; Prokaryotic expression; Inflammatory factors
Reactogenicity
Myc-tag
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Objective To clone and express the gene encoding Schistosoma japonicum myophilin-like protein (SjcMLP) and to study the antigenicity of the recombinant protein. Methods The SjcMLP gene was amplified by PCR. The PCR product was cloned into T vector, and then subcloned into expression vector pQE30. The recombinant plasmid of pQE30-SjcMLP was transformed into E.coli M15, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analysed by SDS-PAGE. The recombinant protein (reSjcMLP)was purified with the Ni-NTA resin, and analysed with SDS-PAGE and Western blot. The titers of sera from C57BL/6 mice immunized subcutaneously with reSjcMLP were detected by ELISA. Results The results of SDS-PAGE and Western blot showed that the molecular weight of expressed fusion protein was around 24.8 kDa and was recognized by the sera from the mice infected with Schistosoma japonicum. The purified protein of reSjcMLP was coated for ELISA test and the IgG titers in the sera from the mice immunized with reSjcMLP were as high as 1∶12 800 reacted with. However, no significant difference was found in worm reduction rates between the immunized mice and control mice. Conclusions The fused recombinant protein of reSjcMLP is successfully ex-pressed and purified. The recombinant protein in this experiment fails to induce significant protection against the challenge infection in C57BL/6 mice.
Antigenicity
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Polyclonal antibodies
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