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首页> 外文期刊>Journal of immunotherapy >Development of a polynucleotide vaccine from melanoma antigen recognized by T cells-1 and recombinant protein from melanoma antigen recognized by T cells-1 for melanoma vaccine clinical trials.
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Development of a polynucleotide vaccine from melanoma antigen recognized by T cells-1 and recombinant protein from melanoma antigen recognized by T cells-1 for melanoma vaccine clinical trials.

机译：从T细胞-1识别的黑素瘤抗原开发多核苷酸疫苗，并从T细胞-1识别的黑素瘤抗原开发重组蛋白用于黑素瘤疫苗临床试验。

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MART-1, a melanoma antigen recognized by T cells-1, is a melanocyte lineage-differentiation antigen expressed only in melanocytes and melanoma cells. This protein is recognized by many T-lymphocyte lines that are human leukocyte antigen (HLA)-A2 restricted and melanoma reactive. These observations have culminated in an array of clinical trials of MART-1 immunization using recombinant viruses or MART-1 immunodominant peptides. Polynucleotide immunization is a promising alternative to recombinant viral vaccines that allows delivery of the full-length cDNA encoding all potential peptide epitopes in a vector that is uncompromised by anti-viral immunity. In preparation for a phase I clinical trial of MART-1 polynucleotide immunization in patients with resected melanoma who were at significant risk for recurrence, the authors constructed a plasmid DNA encoding the MART-1 cDNA under transcriptional regulatory control of the cytomegalovirus immediate early promoter-enhancer and partially deleted intron A. This plasmid directs high-level MART-1 expression in transduced myoblasts and maturing myocytes diffusely throughout the cytoplasm. Immunization of mice with this construct by intramuscular injection elicited MART-1-specific immune responses in all animals. Previous trials of MART-1 immunization have been unable to examine the humoral immune response to MART-1 because of a lack of sufficient, highly purified protein. We have produced and purified Escherichia coli recombinant MART-1 protein using a glutathione-S-transferase fusion protein expression system. Protein staining of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a band of MART-1 protein at approximately 20 kD; and Western immunoblotting with an anti-MART-1 monoclonal antibody confirmed a doublet at approximately 20 kD. These findings are consistent with previous reports using different expression systems for recombinant MART-1. This protein preparation functioned well in enzyme-linked immunosorbent assays (ELISAs) to detect anti-MART-1 antibody responses in a mouse model; and a panel of healthy donor human sera showed minimal binding to ELISA plates coated with the protein, supporting its utility in monitoring human anti-MART-1 antibody responses. The glutathione-S-transferase fusion method yielded approximately 200 micrograms MART-1 per 2-L bacterial culture, enough to coat 100 ELISA plates.
机译：MART-1是被T细胞-1识别的黑色素瘤抗原，是仅在黑色素细胞和黑色素瘤细胞中表达的黑色素细胞谱系分化抗原。该蛋白质被人类白细胞抗原（HLA）-A2限制和黑色素瘤反应性的许多T淋巴细胞系所识别。这些观察结果达到了使用重组病毒或MART-1免疫显性肽进行MART-1免疫的一系列临床试验的结果。多核苷酸免疫是重组病毒疫苗的一种有前途的替代方法，它允许在不受抗病毒免疫影响的载体中递送编码所有潜在肽表位的全长cDNA。在准备将MART-1多核苷酸免疫的黑色素瘤患者复发的重大风险的I期临床试验中，作者构建了在巨细胞病毒即时早期启动子-转录调控下编码MART-1 cDNA的质粒DNA。增强子和部分缺失的内含子A。此质粒指导转导的成肌细胞中的高水平MART-1表达，成熟的心肌细胞分散在整个细胞质中。通过肌内注射用该构建体免疫小鼠在所有动物中引起了MART-1特异性免疫反应。由于缺乏足够的高度纯化的蛋白质，以前的MART-1免疫试验无法检查对MART-1的体液免疫反应。我们使用谷胱甘肽-S-转移酶融合蛋白表达系统生产和纯化了大肠杆菌重组MART-1蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的蛋白质染色显示约20 kD的MART-1蛋白带。用抗MART-1单克隆抗体进行的Western blotting证实了大约20 kD的双链体。这些发现与先前针对重组MART-1使用不同表达系统的报道一致。这种蛋白质制剂在酶联免疫吸附测定（ELISA）中能很好地发挥功能，以检测小鼠模型中的抗MART-1抗体反应。一组健康的供体人血清显示与包被该蛋白的ELISA板的结合极少，支持其在监测人抗MART-1抗体反应中的效用。谷胱甘肽-S-转移酶融合方法每2升细菌培养产生约200微克MART-1，足以覆盖100个ELISA板。

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