en Life Science Life Science مقاله پژوهشی Original Research Article <div style="text-align: justify;"><span style="line-height:2;"><span style="font-family:Times New Roman;"><span style="font-size:14px;"><b><span style="color:black">Background and Objectives:</span></b><span style="color:black">&nbsp;Tenascin C (TNC) and tyrosine-protein phosphatase receptor (PTPRZ-1) are highly expressed in glioblastoma multiforme (GBM) compared to normal brain tissue, making them potential targets for immunotherapy. This study aimed to clone, express, and purify a recombinant multi-domain vaccine candidate combining TNC and PTPRZ-1 for potential application in GBM immunotherapy.</span><br> <b><span style="color:black">Materials and Methods:</span></b><span style="color:black">&nbsp;The target genes were amplified as a double construct (PTPRZ-1 and TNC) from a previously designed triple construct (IL-13R&alpha;-2, TNC, and PTPRZ-1). The pET-28a plasmid was extracted from <i>E. coli</i> TOP10 using the alkaline lysis method. Plasmid purification was verified by PCR with specific primers. Enzymatic digestion of the insert and pET-28a plasmid was performed with restriction enzymes, followed by ligation. Recombinant plasmids were transformed into <i>E. coli</i> TOP10 and <i>E. coli</i> BL21 cells. Confirmed colonies were cultured on a large scale, and recombinant protein expression was induced with IPTG. Purification was performed using Ni‑NTA affinity chromatography under native and denatured conditions. Protein expression was examined using SDS-PAGE and Western blotting, and protein concentration was determined by Bradford assay.</span><br> <b><span style="color:black">Results:</span></b><span style="color:black">&nbsp;Successful cloning and expression of the recombinant PTPRZ-1-TNC construct were confirmed. The purified recombinant protein showed the expected molecular weight (~45 kDa) and was verified by Western blot analysis. The yield was approximately 168&ndash;492 &micro;g per 450 &micro;L elution fraction, as determined by Bradford assay.</span><br> <b><span style="color:black">Conclusion:</span></b><span style="color:black">&nbsp;The cloning, expression, and purification of the recombinant proteins were successfully performed using standard laboratory methods, providing a foundation for future preclinical studies to evaluate their immunogenicity and therapeutic efficacy in GBM immunotherapy. </span></span></span></span><br> &nbsp;</div> Tenascin C, PTPRZ-1, Glioblastoma, Immunotherapy, Vaccine 56 56 http://journal.zums.ac.ir/browse.php?a_code=A-10-3329-5&slc_lang=en&sid=1 Maedeh Parchianlou mparchianloo@gmail.com 5200319475328460092225 5200319475328460092225 No Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran Taravat Talebi talebitaravat@gmail.com 5200319475328460092226 5200319475328460092226 No Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran Neda Mohsen-Pour neda2363@gmail.com 5200319475328460092227 5200319475328460092227 No Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran Ali Sharafi alisharafi@zums.ac.ir 5200319475328460092228 5200319475328460092228 Yes Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran