Anti-CD25 monoclonal antibody enhances the protective efficacy of Schistosoma japonicum GST vaccine via inhibition of CD4+CD25+Foxp3+ regulatory T cells

February 15, 2022 0 Comments

The effect of anti-CD25 monoclonal antibody (anti-CD25 mAb) on the protection efficacy of Schistosoma japonicum 26 kDa GST (glutathione-S-transferase) vaccine was evaluated. Mice were immunized with GST before infection with S. japonicum cercariae and then injected with anti-CD25 mAb. The worm reduction rate was promoted from 24.18% in mice with GST immunization to 47.09% in mice with GST plus anti-CD25 mAb.
Compared with the control group, the percentages of splenic CD4+CD25+Foxp3+ regulatory T cells (Tregs) were significantly lower after administration of anti-CD25 mAb; meanwhile, elevated levels of IFN-γ and IL-2 were secreted by splenocytes. These results indicate that the poor protective efficacy of the GST vaccine against S. japonicum results from the presence of CD4+CD25+Foxp3+ Tregs, while anti-CD25 mAb can partially block CD4+CD25+Foxp3+ Tregs and thus enhance the protective efficacy of the GST vaccine.

Production of polyclonal antibody against HBD-2 by immunization with recombinant GST-HBD-2 fusion protein

For the purpose of detecting the HBD-2 expression at protein level, the recombinant prokaryotic expression vector pGEX-1lambdaT-HBD-2 was constructed and the E. coli-based product of GST-HBD-2 fusion protein was prepared. When rabbit was immunized with the fusion protein, the anti-serum against HBD-2 was produced.
After caprylic acid and ammonium sulfate precipitation, high titer of specific polyclonal antibody against HBD-2, which was detected by ELISA and Western blot, was obtained. This result suggests that recombinant peptide fusion protein could be used instead of the conjugate of peptide-albumin or peptide-thyroid globulin to produce antibody. The obtained antibodies could be used for revealing the tissue distribution of HBD-2 and the regulation of its gene expression.

An alternative easy method for antibody purification and analysis of protein-protein interaction using GST fusion proteins immobilized onto glutathione-agarose

Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromises the interaction between the protein and its target. In these cases, glutathione-S-transferase (GST) is a valuable tag providing a long arm that makes the bait protein accessible to the mobile flow phase of the chromatography.
In the present report, we used a GST fusion version of the 8-kDa protein serine protease inhibitor Kazal-type 3 (SPINK3) as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione-agarose beads was modified from previously reported protocols by using an alternative bifunctional cross-linker (dithiobis(succinimidyl propionate)) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the reuse of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as the bait.

Use of in vivo biotinylated GST fusion proteins to select recombinant antibodies

Over the last 20 years, continuous advances in the field of molecular biology have led to the development of new strategies to discover and produce monoclonal antibodies, notably by phage display.Here we describe a simple procedure for antibody selection that reduces considerably the undesired selection of non-specific antibodies, based on the use of biotinylated GST proteins fused to a target antigenic sequence. This procedure was tested on a collection of 7 different targets and resulted in the selection of a high percentage (71%) of antibodies specific for each target. This simple and effective in vitro procedure has a strong potential to replace animal immunization for the development of specific antibodies.

An antibody-free strategy for screening putative HDM2 inhibitors using crude bacterial lysates expressing GST-HDM2 recombinant protein

Targeting the interaction of p53 with its natural inhibitor MDM2 by the use of small synthetic molecules has emerged as a promising pharmacological approach to restore p53 oncosuppressor function in cancers retaining wild-type p53. The first critical step in the experimental validation of newly synthesized small molecules developed to inhibit MDM2-p53 interaction is represented by the evaluation of their efficacy in preventing the formation of the MDM2-p53 complex. This can be achieved using the in vitro reconstructed recombinant MDM2-p53 complex in cell-free assays.
A number of possible approaches have been proposed, which are however not suitable for screening large chemical libraries, due to the high costs of reagents and instrumentations, or the need of large amounts of highly pure recombinant proteins. Here we describe a rapid and cheap method for high-throughput screening of putative inhibitors of MDM2-p53 complex formation–based on the use of GST-recombinant proteins–that does not require antibodies and recombinant protein purification steps from bacterial cell lysates.

Expression and purification of GST-CML28 fusion protein and preparation of its polyclonal antibody

This study was aimed to investigate the expression of GST-CML28 in Escherichia Coli and to prepare its antibody. The constructed recombinant expression vectors CML28-pGEX-3X were transformed into Escherichia Coli BL21 under IPTG induction. The protein was abstracted from the transformers, and purified by a GSTrap FF column. The rabbits were immunized by the purified fusion protein to produce serum with anti-CML28 antibody.
The serum was purified by chromatographic column stuffed with glutathione Sephamse 4B to get the antibody. The specific antibody against CML28 was further identified by ELISA, Western blot, immunohistochemistry and quantum dot luminescence. The results indicated that GST-CML28 fusion protein was expressed in Escherichia coli and its specific polyclonal antibody was obtained. It is concluded that the anti-CML28 polyclonal antibodies with high titer and specificity are successfully prepared. These antibodies provide an useful experimental tool to profoundly research the physiological significance and biological function of the CML28 gene.

Recombinant GST-I-A beta 28-induced efficient serum antibody against A beta 42

Six recombinant proteins GST-A beta 28/A beta 35/A beta 42 and GST-I-A beta 28/A beta 35/A beta 42 [I was the abbreviation for an immunostimulatory sequence that consisted of pan HLA DR binding epitope (PADRE) and Tetanus toxin epitope (TT)] were used as antigens after expressed and purified to immunize mice. The strongest antibody response against A beta 42 (titer 1:3200) was achieved by GST-I-A beta 28 or GST-A beta 42 immunization. However, IgG1 and IgG2b were the predominant serum antibody isotype responses by GST-I-A beta 28 immunization, whereas did IgG2a by GST-A beta 42 immunization.

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Thus, it indicated that GST-I-A beta 28 immunization in a mouse mainly evoked a stronger Th-2-type response; whereas, GST-A beta 42 immunization mainly elicited a Th-1-type response. Moreover, GST-I-A beta 28-induced serum antibodies had higher specificity to A beta 42 monomers and oligomers than to protofibrils and mature fibrils and exhibited the highest efficacy to block A beta 42 aggregation or fibrillogenesis and to disassemble A beta 42 aggregates in vitro. GST-I-A beta 28-induced serum antibodies also showed the most protective and restorative effects on target cells in vitro by inhibiting or neutralizing A beta 42-induced cytotoxicity. All of the above results indicated that A beta 28 could be speculated to substitute for A beta 42 and would become a better antigenic peptide for Alzheimer’s disease immunotherapy in the presence of additional Th-cell epitopes such as the immunostimulatory sequence (I) applied in this study.

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