Construction of a fusion expression plasmid containing the G250 gene and human granulocyte-macrophage colony stimulating factor and its significance in renal cell carcinoma

Abstract

Abstract. This study aimed to construct a eukaryotic expression plasmid containing the G250/MN/CA IX (G250) and human granulocyte-macrophage colony stimulating factor (hGM-CSF) genes, and to detect the expression of these proteins in vitro by recombinant plasmids in eukaryotic cells. pORF-hGM-CSF and pcDNA3.0-G250 were used as the template to amplify G250 and hGM-CSF by routine polymerase chain reaction (PCR). The two PCR products were cloned into the eukaryotic vector pVAX1, in order to construct a recombinant plasmid pVAX1-G250-hGM, and the plasmid was transfected into human embryonic kidney 293 cells. The protein expression was then determined by immunocytochemistry, atomic force microscopy, ELISA and Western blotting. DNA sequencing showed that the cloned G250 and hGM-CSF sequences were consistent with the reported Gene Bank ones. Moreover, a high expression was noted following recombinant plasmid transfection of the G250 and hGM-CSF proteins. Thus, the eukaryotic expression vector pVAX1-G250-hGM containing G250 and hGM-CSF was constructed, allowing for the investigation of the anti-G250 antigen vaccine and immune response mechanisms of biological immunotherapy in renal cell carcinoma. IntroductionRenal cell carcinoma (RCC) is a common malignant tumor of the urinary system. One out of nearly a million individuals succumb to the disease each year worldwide and the incidence is on the increase (1). RCC patients frequently present with subclinical disease and 20-30% of patients are admitted (2).However, traditional radical nephrectomy in RCC in the early stage has a positive effect, which is not the case in advanced stage and metastatic RCC. Simultaneously, RCC is not sensitive to radiotherapy and chemotherapy (3). Since RCC is a highly immunogenic tumor, advances in molecular and immune biology allow for the potential use of tumor vaccines as immune therapy (4). G250/MN/CA IX (MN antigen receptor/ carbonic anhydrase-9) is one of the tumor markers that possess favorable tumor specificity (5). The specific expression of G250/MN/CA IX in RCC renders it a key target for cancer diagnosis and treatment. In this study, a eukaryotic expression vector, containing the G250/MN/CA IX and human granulocyte-macrophage colony stimulating factor (hGM-CSF) genes, was constructed and transfected into human embryonic kidney 293 (HEK 293) cells. The fusion protein expression and immunoreactivity were then detected to establish anti-renal cell carcinoma vaccines for studies based on the G250 gene. Materials and methodsMaterials. E. coli Top 10, vector pVAX1, recombinant plasmid pORF-hGM-CSF and pcDNA3.0-G250 were obtained from

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<p>Loss of <em>KCNJ15</em> expression promotes malignant phenotypes and correlates with poor prognosis in renal carcinoma</p>

“…Renal cell carcinoma (RCC), accounting for 80%–90% of the renal tumors, is a common malignant tumor of the genitourinary system 1,2. Approximately 75%–85% of the RCC cases are clear cell RCC (ccRCC) 3,4.…”

“…Around one-third of the patients diagnosed with RCC already have locally advanced or metastatic disease. The metastasis of RCC is the leading cause of the high mortality rate in patients with RCC 2,5,6. Therefore, the study of the molecular mechanism of the occurrence, development, and metastasis of RCC and the identification of new specific early diagnostic biomarkers and therapy targets for RCC are imperative.…”

Combining DNA Vaccine and AIM2 in H1 Nanoparticles Exert Anti-Renal Carcinoma Effects via Enhancing Tumor-Specific Multi-functional CD8+ T-cell Responses

“…CAIX shows apparent tumor specificity and could serve as an ideal target for immunotherapy of renal carcinoma (13). So far, there have been many studies for CAIX antigen-based renal carcinoma vaccine, including peptide vaccine, CAIX antigen-loading dendritic cells (DC), and DNA vaccine (14,15). Remarkable progress has been observed in the development of vaccines, but the therapeutic effect and safety problem remain challenging (16).…”