Departamento de Histología, Facultad de Medicina, Universidad Autónoma de Nuevo León (UANL), Monterrey, N,L,, México.
Microb Cell Fact. 2008 Jul 28;7:22. doi: 10.1186/1475-2859-7-22.
Chemokines are a large group of chemotactic cytokines that regulate and direct migration of leukocytes, activate inflammatory responses, and are involved in many other functions including regulation of tumor development. Interferon-gamma inducible-protein-10 (IP-10) is a member of the C-X-C subfamily of the chemokine family of cytokines. IP-10 specifically chemoattracts activated T lymphocytes, monocytes, and NK cells. IP-10 has been described also as a modulator of other antitumor cytokines. These properties make IP-10 a novel therapeutic molecule for the treatment of chronic and infectious diseases. Currently there are no suitable live biological systems to produce and secrete IP-10. Lactococcus lactis has been well-characterized over the years as a safe microorganism to produce heterologous proteins and to be used as a safe, live vaccine to deliver antigens and cytokines of interest. Here we report a recombinant strain of L. lactis genetically modified to produce and secrete biologically active IP-10.
The IP-10 coding region was isolated from human cDNA and cloned into an L. lactis expression plasmid under the regulation of the pNis promoter. By fusion to the usp45 secretion signal, IP-10 was addressed out of the cell. Western blot analysis demonstrated that recombinant strains of L. lactis secrete IP-10 into the culture medium. Neither degradation nor incomplete forms of IP-10 were detected in the cell or supernatant fractions of L. lactis. In addition, we demonstrated that the NICE (nisin-controlled gene expression) system was able to express IP-10 "de novo" even two hours after nisin removal. This human IP-10 protein secreted by L. lactis was biological active as demonstrated by Chemotaxis assay over human CD3+T lymphocytes.
Expression and secretion of mature IP-10 was efficiently achieved by L. lactis forming an effective system to produce IP-10. This recombinant IP-10 is biologically active as demonstrated by its ability to chemoattract human CD3+ T lymphocytes. This strain of recombinant L. lactis represents a potentially useful tool to be used as a live vaccine in vivo.
趋化因子是一大类趋化细胞因子，可调节和指导白细胞的迁移、激活炎症反应，并参与许多其他功能，包括调节肿瘤的发展。干扰素-γ诱导蛋白-10（IP-10）是趋化因子家族细胞因子的 C-X-C 亚家族的成员。IP-10 特异性趋化激活的 T 淋巴细胞、单核细胞和 NK 细胞。IP-10 也被描述为其他抗肿瘤细胞因子的调节剂。这些特性使 IP-10 成为治疗慢性和传染性疾病的新型治疗分子。目前，没有合适的活生物系统来生产和分泌 IP-10。多年来，乳酸乳球菌已被很好地描述为一种生产异源蛋白的安全微生物，并可作为一种安全的活疫苗，用于传递感兴趣的抗原和细胞因子。在这里，我们报告了一种经过基因改造的重组乳酸乳球菌菌株，该菌株可生产和分泌具有生物活性的 IP-10。
从人 cDNA 中分离出 IP-10 编码区，并在 pNis 启动子的调控下克隆到乳酸乳球菌表达质粒中。通过与 usp45 分泌信号融合，IP-10 被定位到细胞外。Western blot 分析表明，重组乳酸乳球菌菌株将 IP-10 分泌到培养基中。在乳酸乳球菌的细胞或上清液部分均未检测到 IP-10 的降解或不完全形式。此外，我们证明 NICE（乳链菌肽控制基因表达）系统甚至在乳链菌肽去除后两个小时仍能“从头开始”表达 IP-10。这种由乳酸乳球菌分泌的人 IP-10 蛋白具有生物活性，正如通过趋化性测定对人 CD3+T 淋巴细胞的作用所证明的那样。
通过乳酸乳球菌有效地实现了成熟 IP-10 的表达和分泌，形成了一种有效的生产 IP-10 的系统。这种重组 IP-10 具有生物活性，能够趋化人 CD3+T 淋巴细胞。这种重组乳酸乳球菌菌株代表了一种潜在有用的工具，可以作为活疫苗在体内使用。
