Prokaryotic Expression of DJ-1 Gene of Chicken and Preparation of Anti-serum Against DJ-1 Protein
0
Citation
0
Reference
20
Related Paper
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
Cite
To clone and express C31B8.8 gene of wild-type Caenorhabditis elegans, and study the immunological characteristic of the recombinant protein.Total RNA was extracted from cultivated C. elegans and reversely transcribed into cDNA. C31B8.8 gene was amplified by PCR and cloned into pMD-18T vector for sequencing. The accurate sequence was subcloned into the expression vector pET-30a with (His) 6-tag. The recombinant plasmid was transformed into E. coli BL21 and followed by expression of the protein induced by IPTG. The recombinant protein was identified by using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and Western blotting 10 BALB/c mice were randomly divided into C31B.8-immunized group and PBS + adjuvant group Mice in C31B8.8-immunized group were immunized with 40 microg of purified C31B8.8 antigen formulated in Freund's adjuvant Mice in PBS + adjuvant group received only adjuvant emulsified with PBS. All the mice received four immunizations every week with the same dose of antigen. Serum samples were collected at pre-immunization and certain time after immunization and the antibody titer was analyzed by ELISA. The recombinant C31B8.8 protein and soluble components of Angiostrongylus cantonensis fourth-stage larvae were identified by Western blotting.The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing. MALDI-TOF-MS and Western blotting analysis showed that the recombinant C31B8.8 protein was the target protein. Compared with PBS + adjuvant group, mice immunized with purified protein C31B8.8 produced higher level of IgG. The anti-C31B8.8 serum recognized recombinant C31B8.8 protein, and reacted with soluble antigens of A. cantonensis fourth-stage larvae.C elegans C31B8 gene shows certain immunogenicity and immunoreactivity, and the soluble antigens of A. cantonensis fourth-stage larvae can react with anti-C31B8.8 serum.
Cite
Citations (0)
To express hCacyBP in E.coli and prepare mouse polyclonal antibody against hCacyBP so as to study tissue distribution of hCacyBP and evaluate its role during formation of multidrug resistance to gastric cancer.hCacyBP gene was subcloned into an expression vector pET28a, and then the recombinant vector was transformed into E. coli BL21. The recombinant protein was expressed in BL21 under IPTG induction. Using purified hCacyBP as immunogen, mouse polyclonal antibody against hCacyBP was prepared. hCacyBP was detected by Western blot in 10 kinds of rabbit tissues. Expression and distribution of hCacyBP in SGC7901/VCR and SGC7901 cells were detected by Western blot and immunohistochemical staining.hCacyBP was successfully expressed in E. coli. The Western blot analysis showed that hCacyBP was expressed in all 10 kinds of rabbit tissues, but expression of brain and liver tissues were higher as compared with other tissues. Expression and distribution of hCacyBP in both SGC7901/VCR and SGC7901 cells had no significant difference.CacyBP expressed widespreadly in varied tissues. Polyclonal antibody against hCacyBP that we prepared has high specificity, which provides a powerful tool for studying the function of hCacyBP.
Polyclonal antibodies
Immunogen
Cite
Citations (2)
The aim of this work was the in vitro expression of the recombinant fusion (F) protein of Newcastle disease virus (NDV). The pBT7-N-His-Fusion-NDV expression plasmid which carries the recombinant F protein encoding gene from local Indonesian isolates, was prepared and transformed into E. coli BL21 (DE3). To detect bacterial colonies carrying the recombinant plasmid, a restriction endonuclease analysis was performed using the EcoRI restriction endonuclease. These results showed that the pBT-N-His-Fusion-NDV plasmid was successfully isolated with a size of 4.601 bp, and three recombinant plasmids carrying the gene coding for the recombinant F protein of NDV were obtained. Selected recombinant plasmids were then in vitro by using a cell-free protein expression system followed by visualization of the recombinant F protein on a 12% SDS-PAGE gel both by Coomassie Brilliant Blue staining and Western blotting. Recombinant F protein was successfully in vitro expressed by using a cell-free protein expression system as indicated by a specific single protein band with a molecular mass of 25.6 kDa.
Cite
Citations (2)
Introduction: Saponaria officinalis have various saponin isoforms. Saponin is a ribosome-inactivating protein (RIP). The SO9 isoform of saponins depurinates the adenine 4324 in the preserved GAGA sequence resulting in impairment of protein production. In this study, the S09 isoform was expressed in E. coli and its antibody titers were evaluated in Mouse. Methods: The S09 gene was synthesized and isolated from the pUC57-S09 recombinant plasmid using the restriction enzymes BamH1 and Sal1, and then cloned in the expression vector pET28a(+). Expression of the new recombinant protein was induced by IPTG. The recombinant S09 protein was purified by Ni affinity chromatography. The recombinant protein was confirmed through western blotting. The Mouse were vaccinated through intraperitoneal injection of the purified protein and serum IgG titer was measured through ELISA.Results: Subcloning of S09 gene in the pET28a(+) expression vector was confirmed by PCR and enzymatic digestion. The presence of 29 kDa protein band in SDS-PAGE showed the high expression of recombinant protein. The recombinant S09 protein was detected by polyclonal antibody. After injection of the protein to the test groups, the antibody titer was measured by ELISA. Conclusion: The adjuvant property and immunogenicity of the purified recombinant S09 antigen showed that this antibody can be used to detect the presence of S09 in Saponaria officinal is, as a candidate for vaccine, for production of diagnostic kits, and in human cells anticancer studies.
Subcloning
Polyclonal antibodies
Myc-tag
KDEL
Cite
Citations (0)
To clone and express the aegyptin-like protein (alALP) encoding gene from Aedes albopictus salivary gland, and analyze its antigenicity.The homology, secondary structure and antigen peptides of alALP and aegyptin protein (GenBank No. ABF18122.1) was analyzed by bioinformatics software tools. Total RNA was extracted from Ae. albopictus salivary gland. The coding region of alALP (GenBank No. AY826121) was amplified by PCR. RT-PCR product was digested with restriction enzyme and ligated into a pGEX-6P-1 vector. The recombinant pGEX-6P-1-alALP plasmid was transformed into E. coli BL21 and induced by IPTG. The recombinant soluble GST-alALP fusion protein was purified with Glutathione Sepharose 4B. The expression product was analyzed by SDS-PAGE and Western blotting. Mice were immunized each with 60 microg purified GST-alALP at every 2 weeks for 3 times, and mouse anti-GST-alALP serum was prepared. Western blotting assay with mice anti-GST-alALP serum and serum of mice exposed to Ae. albopictus bites was used to analyze its antigenicity.Bioinformatics prediction results showed that alALP and aegyptin had 65.58% homology with a similar secondary structure, and a conservative polypeptide. The product of RT-PCR was 762 bp. The recombinant plasmid pGEX-6P-1-alALP was confirmed by double restriction enzyme digestion, PCR and sequencing. SDS-PAGE and Western blotting analysis showed that the bacteria containing recombinant plasmid pGEX-6p-1-alALP expressed a soluble recombinant fusion protein (M(r) 56 000) after being induced with IPTG. Western blotting analysis revealed that GST-alALP protein was recognized by mouse anti-GST-alALP serum and serum of mice ex- posed to Ae. albopictus bites.Mature peptide gene of alALP can be expressed in prokaryotic expression system, and the recombinant protein shows antigenicity.
Antigenicity
Cite
Citations (0)
To clone and express a novel protein analogous to TIP (T cell immunomodulatory protein) of Plasmodium berghei ANKA and prepare its polyclonal antibody.The PbTIP encoding nucleotide sequence was searched from the Plasmodium berghei genomic database and amplified by PCR. The gene was sub-cloned into prokaryotic expression vector pGEX4T-1 and expressed in E.coli BL21 (DE3). After induction by IPTG, the expression of PbTIP-GST fusion protein was characterized by SDS-PAGE and Western blotting. The inclusion bodies of GST-PbTIP fusion protein were injected into BALB/c mouse. Anti-sera were identified by indirect fluorescent antibody test and western blotting.The PbTIP-GST fusion protein was successfully expressed in the form of inclusion bodies, by controlling the temperature and concentration of IPTG. Anti-PbTIP-GST sera were acquired with high titer. The sera specifically recognized the PbTIP with a band of 60 000 in P.berghei infected erythrocyte protein.PbTIP/GST fusion protein and polyclonal antibody have been obtained.
Polyclonal antibodies
Plasmodium berghei
clone (Java method)
Cite
Citations (0)
To induce the expression of F10 in E.coli and prepare the rabbit polyclonal antibody against it.The gene of F10 was amplified by PCR, and cloned into expression vector pET-GST to construct recombinant expression plasmid pET-GST/F10. The recombinant plasmid was transformed into E.coli BL21 and induced to express recombinant protein with IPTG. The fusion protein was further purified by affinity chromatography and analyzed by SDS-PAGE and Western blot. A rabbit was immunized with the purified F10 fusion protein to produce polyclonal antibody, and the production of antibody was confirmed by ELISA.Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid contained correct coding region of F10. SDS-PAGE demonstrated that the recombinant protein was expressed with the expected molecular weight at 61 kD. After purified, the purity of the fusion protein was above 90%. Western blot confirmed the recombinant protein was GST/F10 fusion protein. Rabbit polyclonal antibody was obtained, the titer of which was 1:20 000.F10 recombinant expression vector has been successfully constructed and F10 protein has been expressed. The obtained rabbit anti F10 antibody has a high titer and will facilitate the study of the biological function of F10.
Polyclonal antibodies
Cite
Citations (0)
Objective To construct a recombinant vector of sperm-specific human lactate dehydrogenase ( hLDH-C4 ), express it in Escherichia coli ( E. coli ) BL21 ( DE3 ) and utilize it in the detection of anti-sperm antibody. Methods The coding sequence of hLDH-C4 was amplified from human testis λTripIEx cDNA library, and inserted into pET-28a( + ) after restriction enzyme digestion with Hind Ⅲ and Xho Ⅰ. The resultant recombinant vector was used to transform E. coli BL21 ( DE3 ) and the His-Tag fused hLDH-C4 was expressed after induction with IPTG. Western blot was used to analyzed the recombinant protein and LDH activity of bacterial lysates was determined. An indirect ELISA method for the detection of anti-sperm antibody was established by using the recombinant hLDH-C4 as antigen matrix. Results pET-28a( + )-hLDHC was successfully established. The protein with size of 35kD could be induced by IPTG when the recombinant plasmid was transfected into E. coli BL21 ( DE3 ). Western blot showed that the recombinant protein could be specifically recognized beth by anti-His tag monoclonal antibody and by rabbit anti-human LDH-C4 antibody. In addition, the recombinant protein showed high-level LDH activity when the bacterial lysate after IPIG induction was used to check LDH activity. The recombinant hLDH-C4 was confirmed when it was used in indirect EL1SA to detect anti-hLDH-C4 antibody. Conclusions The coding sequence of hLDH-C4 is cloned into the vector pET-28a( + ) and recombinant hLDH-C4 was expressed at a high level in E. coli. The recombinant hLDH-C4 is useful in the detection of anti-sperm antibody.
Key words:
L-lactate dehydrogenase; Isoenzyme; Spermatozoa; Autoantibodies; Escherichia coli; Recombinant proteins
Cite
Citations (0)
To clone and express a high mobility group box 1(HMGB1) protein of Schistosomajaponicum (Mainland strain) and analyze its function.The DNA fragment of open reading frame encoding Sj HMGB 1 protein was amplified by RT-PCR from the mRNA of S. japonicum worms, then it was subcloned into the expression vector pET28a(+) to form the recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21(DE3), and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombinant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding capacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT-PCR. Female ICR mice were immunized with the recombinant SjHMGB1 protein and infected with 45 +/- 2 cercariae of S. japonicum after three immunizations. Forty-two days post-infection, the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue, respectively. The worm and egg reduction rates were calculated respectively.A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT-PCR, which was the open reading frame (ORF) encoding SjHMGBlprotein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1-pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a(+). The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG, and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA, and the recombinant protein immunized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum-infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT-PCR and Western blot results showed that SjHMGB1 was abundantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effective immune protection against S. japonicum.The gene encoding HMGB1 from S. japonicum and the soluble recombinant SjHMGB1 protein with natural functional activity are obtained, and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.
Cloning (programming)
Cite
Citations (1)