RECOMBINANT MYCOBACTERIUM-BOVIS BCG SECRETING FUNCTIONAL INTERLEUKIN-2 ENHANCES GAMMA-INTERFERON PRODUCTION BY SPLENOCYTES
RECOMBINANT MYCOBACTERIUM-BOVIS BCG SECRETING FUNCTIONAL INTERLEUKIN-2 ENHANCES GAMMA-INTERFERON PRODUCTION BY SPLENOCYTES
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DOI：
10.1128/iai.62.6.2508-2514.1994
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发表时间：
1994-06-01
影响因子：
3.1
通讯作者：
DEWOLF, WC
中科院分区：
文献类型：
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作者：
ODONNELL, MA;ALDOVINI, A;DEWOLF, WC
牛分枝杆菌卡介苗基因工程表达和分泌小鼠白细胞介素-2（IL-2）和大鼠IL-2。将编码IL-2的基因插入到BCG HSP 60启动子控制下的大肠杆菌-BCG穿梭质粒中。为了便于研究在该系统中产生的蛋白质，IL-2基因产物（i）单独表达，（ii）在氨基末端与分枝杆菌α-抗原分泌信号序列一起表达，（iii）在氨基末端与流感病毒血凝素表位标签一起表达，以及（iv）与分泌信号序列和表位标签一起表达。当用α-抗原信号序列表达时，生物活性IL-2分泌到细胞外培养基中。细胞内IL-2和细胞外IL-2的蛋白质印迹（免疫印迹）分析显示，分泌信号在分泌时从重组淋巴因子适当地切割。为了评估重组BCG刺激脾细胞群中细胞因子产生的能力，将小鼠脾细胞与野生型或产生IL-2的BCG一起培养。分泌IL-2的BCG克隆刺激γ干扰素产生的大幅增加，这可以通过向BCG中加入外源性IL-2来再现。IL-6、IL-10、肿瘤坏死因子α和粒细胞-巨噬细胞集落刺激因子的水平没有显著变化，而IL-4和IL-5仍然检测不到（小于50 pg/ml）。分泌IL-2的BCG刺激后γ干扰素的产生增加，与菌株无关。表达哺乳动物细胞因子的重组BCG提供了递送细胞因子的新手段，并且可以增强BCG在免疫和癌症治疗中的免疫刺激特性。
Mycobacterium bovis BCG was genetically engineered to express and secrete mouse interleukin-2 (IL-2) and rat IL-2. Genes encoding IL-2 were inserted into an Escherichia coli-BCG shuttle plasmid under the control of the BCG HSP60 promoter. To facilitate study of proteins produced in this system, the IL-2 gene product was expressed (i) alone, (ii) with the mycobacterial alpha-antigen secretion signal sequence at the amino terminus, (iii) with an influenza virus hemagglutinin epitope tag at the amino terminus, and (iv) with both the secretion signal sequence and the epitope tag. When expressed with the alpha-antigen signal sequence, biologically active IL-2 was secreted into the extracellular medium. Western blot (immunoblot) analysis of the intracellular IL-2 and extracellular IL-2 revealed that the secretion signal was appropriately cleaved from the recombinant lymphokine upon secretion. To assess the ability of recombinant BCG to stimulate cytokine production in a splenocyte population, mouse splenocytes were cultured together with wild-type or IL-2-producing BCG. IL-2-secreting BCG clones stimulated substantial increases in gamma interferon production, which could be reproduced by the addition of exogenous IL-2 to BCG. Levels of IL-6, IL-10, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor were not significantly changed, while IL-4 and IL-5 remained undetectable (less than 50 pg/ml). The enhanced production of gamma interferon in response to IL-2-secreting BCG was strain independent. Recombinant BCG expressing mammalian cytokines provides a novel means to deliver cytokines and may augment the immunostimulatory properties of BCG in immunization and cancer therapy.