Prokaryotic Expression of DJ-1 Gene of Chicken and Preparation of Anti-serum Against DJ-1 Protein
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Abstract:
To investigate the function of DJ-1 gene of chicken , a panel of polyclonal antibodies against DJ-1 was needed. In this paper , the CDS region of DJ-1 from cDNA of DF-1 cells were amplified by PCR and cloned it into pET-32a for prokaryotic expression. The BALB/c mice were immunized with the expressed recombinant protein. The results showed that the recombinant DJ-1 fusion protein was abundant and soluble under 3 h induced with 0.4mmol/L IPTG at 37 ℃. ELISA and Western blot results demonstrated that the anti-serum from mice specifically reacted with the recombinant DJ-1. The sera will support the further study of DJ-1 function in the avian leukosis virus subgroup J infection.Keywords:
Polyclonal antibodies
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[Cloning, expression and immunogenicity analysis of malate dehydrogenase gene of Toxoplasma gondii].
To clone and express the malate dehydrogenase (MDH) gene of Toxoplasma gondii, and analyze the immunogenicity.Total RNA was extracted from tachyzoites of RH strain of T. gondii (GenBank accession No. AY650028). The coding region of TgMDH was amplified with a pair of specific primers. The product of RT-PCR was digested with double restriction enzyme and ligated into pET30a (+) vector. The recombinant pET30a (+)-TgMDH plasmid was transformed into E. coli DH5alpha. The positive clones were confirmed by the double restriction enzyme digestion, PCR and sequencing. The correct plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed proteins were analyzed by SDS-PAGE. Conditions for expression were optimized. Abundant soluble rTgMDH protein was purified with Ni-NTA affinity chromatography. Mice was intranasally immunized with purified rTgMDH and murine anti-rTgMDH serum was prepared. Western blotting with murine anti-rTgMDH serum and rabbit anti-T. gondii serum was used to analyze its immunogenicity.The product of RT-PCR was with 951 bp. The recombinant plasmid pET30a(+)-TgMDH was confirmed by the double restriction enzyme digestion, PCR and sequencing. A soluble recombinant protein with relative molecular weight of 36 000 was analyzed by SDS-PAGE, followed by coomassie blue staining. Western blotting revealed that rTgMDH can be recognized by murine anti-rTgMDH serum and rabbit anti-T. gondii serum.TgMDH gene has been expressed in prokaryotic expression system and shows immunogenicity.
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In order to express a novel gene named as BCL-G(L); of swine in E.coli and prepare its polyclonal antibody.The contig sequence of the gene was predicted and in silicon cloned by blasting the human BCL-G(L); in swine ESTs database in NCBI. The cloning sequence was obtained by RT-PCR from swine spleen. The cloning sequence was identified by sequencing and compared with the contig sequence. Then the gene was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid named as pET32a-BCL-G(L);. The fusion protein pET32a-BCL-G(L); was expressed in E.coli BL21 and purified using a His-tag fusion protein purification kit. Then guinea pigs were immunized with the purified protein to get the specific polyclonal antibody.The titer of the antibody was 1:800 detected by ELISA. The protein BCL-G(L); can be specifically detected by western blot assay using the polyclonal antibody.The novel swine gene BCL-G(L); was cloned and expressed in E.coli and its polyclonal antibody was prepared successfully.
Polyclonal antibodies
Cloning (programming)
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[Objective] The aim of the study was to obtain monoclonal antibody against chicken CD8 molecule.[Method] A fragment of chicken CD8α gene was amplified by PCR with a pair of designed primers.Then two recombinant plasmids containing the amplified fragment were constructed.After prokaryotic expression and purification,the obtained recombinant protein was used to immunize BALb/c mice.Finally,the spleen cells were fused with myeloma cells(SP2/0),and antibody titer of culture supernatant was detected by ELISA.[Result] A 510-bp gene fragment was amplified by PCR.The recombinant plasmid pET-32a-CD8α was transformed into E.coli,and 39 kDa His-CD8α fusion protein was induced to expression.After subcloning,the culture supernatant was detected by ELISA.A hybridoma cell strain,which could stably excrete antibody against CD8α protein,was obtained and named C11.The ELISA titer of cell supernatant was higher than 1:640.[Conclusion] A hybridoma cell strain has been established using CD8α expressed in prokaryotic system as immunogen.
Subcloning
Immunogen
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Objective To prepare monoclonal antibodies (mAbs) against chicken cell cycle checkpoint kinase 2 (cChk2). Methods The cChk2 gene was amplified by reverse transcription PCR (RT-PCR) and subcloned into the prokaryotic expression vector pGEX-4T-3. After induced by IPTG, cChk2 was expressed in BL21 (DE3) E.coli cells and analyzed by SDS-PAGE to determine its soluability. BALB/c mice were immunized with cChk2 protein peritoneally. Indirect immunofluorescence assay (IFA) and Western blotting were used to detect anti-serum; if the detection result was positive, IFA and limited dilution was performed to screen hybridoma clones that produced antibodies against cChk2. Results cChk2 was mainly expressed in inclusion bodies. The anti-sera were able to recognize Chk2. Nine positive hybridoma clones were obtained and identified as 1F4, 2D9, 2G1, 3D9, 3E3, 4B5, 4E2, 5C9 and 5F7. Conclusion The study has prepared mAbs against cChk2 with a good specificity and a high titer.
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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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Aim To express recombinant bovine IL-4 (rBoIL-4) in Escherichia coli and prepare monoclonal antibody (mAb) against rBoIL-4. Methods The IL-4 gene without coding signal peptides was amplified from pSP73-BoIL-4 by PCR, then inserted into prokaryotic expression vector pGEX-6p-1 and pET-30a(+). The recombinant plasmids pGEX-6p-1-BoIL-4 and pET-BoIL-4 were transformed into DH5α for sequencing. After sequencing confirmation, the two recombinant plasmids were transformed into expression bacteria BL21 and BL21(DE3) respectively. BALB/c mice were immunized with the purified protein rHis-BoIL-4. With the purified rGST-BoIL-4 as detecting antigen, mAb-produced hybridoma cells against BoIL-4 were screened by indirect ELISA. The specificity of the mAbs was characterized by indirect ELlSA, Dot-ELlSA and Western blot. Results The recombinant bacteria BL21(pGEX-6p-1-BoIL-4) and BL21(DE3)(pET-BoIL-4) were developed. After induced by IPTG, SDS-PAGE analysis showed that the expression products of rGST-BoIL-4 and rHis-BoIL-4 had a molecular weight of 39 kD and 19 kD respectively, and expressed in inclusion body form. Seven hybridoma cell lines secreting mAbs against BoIL-4, named 2B8, 4A10, 5D6, 5D8, 7G10, 8B7 and 10F8 were obtained. The immunoglobulin subclasses were IgG1. The ascitic titers of these mAbs were 5 000, 16 0000, 10 000, 640 000, 5 000, 40 000 and 40 000, respectively. In Dot-ELISA, all mAbs could only react to the immunogen and the detecting antigen. Western-blot analysis confirmed that mAbs could only react to the corresponding recombinant proteins. The mAbs also reacted to the standard recombinant boIL-4 with biological activity. Conclusion Seven mAbs specific to rBoIL-4 protein are obtained, which may have important application value in further study on diagnosis and pathogenesis of cattle diseases.
Immunogen
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To clone and express the actin gene of Toxoplasma gondii, and analyze the immunoreactivity of the recombinant protein.Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgACT gene was amplified with a pair of specific primers which were designed according to the coding sequence of TgACT gene (Accession No. XM_002369622.1). The RT-PCR product was cloned into the prokaryotic expression pET-30a (+) vector. The recombinant pET30a-TgACT plasmid was transformed into E. coli DH5alpha. The positive clones were selected through the colony-PCR and confirmed by the double restrict enzyme digestion and sequencing. The correct pET30a-TgACT plasmid was transformed into E. coli BL21(DE3) and induced by IPTG. The expressed proteins were analyzed by SDS-PAGE. Western blotting assay was performed with anti-poly-histidine tag (anti-His) antibody or rabbit anti-T. gondii serum.The product of RT-PCR was with 1 100 bp. The recombinant plasmid pET30a-TgACT was confirmed by colony-PCR, double restriction enzyme digestion and sequencing. SDS-PAGE results showed that the target protein was expressed in E. coli BL21(DE3) in the form of inclusion bodies with a rough molecular weight of 49 000. The purified soluble protein was obtained by using denaturation, renaturation and purification. Western blotting revealed that rTgACT can be recognized by anti-His antibody and rabbit anti-T. gondii serum.The recombinant plasmid pET30a-TgACT has been successfully constructed, and the recombinant protein TgACT is produced in E. coli and maintains specific immunoreactivity.
Cloning (programming)
Inclusion bodies
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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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To express esophageal cancer related gene-1 (ECRG-1)--in E. coli.The human ECRG-1 was cloned by RT-PCR. Expression plasmid of the ECRG-1/GST pi fusion gene was constructed and introduced into E. Coli. The fusion protein was induced to express by ITPG. The recombinant protein was identified by Western blot. BALB/C mice were immunized with the protein purified by SDS-PAGE for the preparation of polyclonal antibody.DNA sequencing confirmed that the sequence of ECRG-1 was identical to that of the previously obtained by 5'-RACE technique. Expression of the protein in E. coli was verified by Western blot. The polyclonal antibody obtained from immunized BALB/C mice had a title of 1:3,200.The antibody available is useful for the further study of structure and function of the esophageal cancer related protein.
Polyclonal antibodies
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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; Immunocompetence
Myc-tag
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