یکشنبه 26 مهر 1405
[Archive]
پذیرفته شده و در حال انتشار
برگشت به فهرست مقالات |
برگشت به فهرست نسخه ها
Ethics code: IR.ZUMS.REC.1400.404
چکیده: (18 مشاهده)
Background and Objectives: 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.
Materials and Methods: The target genes were amplified as a double construct (PTPRZ-1 and TNC) from a previously designed triple construct (IL-13Rα-2, TNC, and PTPRZ-1). The pET-28a plasmid was extracted from E. coli 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 E. coli TOP10 and E. coli 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.
Results: 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 168–492 µg per 450 µL elution fraction, as determined by Bradford assay.
Conclusion: 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.
Materials and Methods: The target genes were amplified as a double construct (PTPRZ-1 and TNC) from a previously designed triple construct (IL-13Rα-2, TNC, and PTPRZ-1). The pET-28a plasmid was extracted from E. coli 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 E. coli TOP10 and E. coli 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.
Results: 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 168–492 µg per 450 µL elution fraction, as determined by Bradford assay.
Conclusion: 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.
| بازنشر اطلاعات | |
 |
این مقاله تحت شرایط Creative Commons Attribution-NonCommercial 4.0 International License قابل بازنشر است. |
