Development of blood-brain barrier permeable TAT chimera Interferon-β for the treatment of malignant gliomas.

TAT嵌合体蛋白具有作为蛋白质通过血脑屏障传递的载体的潜力，并能使靶物质通过全身给药到达脑实质。我们研究了使用血脑屏障（BBB）渗透性TAT嵌合体干扰素-β治疗恶性胶质瘤的可能性。我们对人干扰素-β (HuIFN-β)进行了RT-PCR，其中His-tag和tat蛋白转导结构域（PTD）连接在c端。确认DNA序列并将其亚克隆到质粒表达载体上。将所设计的质粒转化到大肠杆菌中，进行了蛋白的大规模表达。TAT-HuIFN-β提取为不溶性组分，用sb3 - 14溶解。TAT-HuIFN-β用Ni基质纯化，蛋白重折叠。作为对照研究，通过腹腔注射TAT-HA肽，与单独注射HA相比，在大鼠脑内检测到高浓度的HA抗原。这一结果支持了TAT嵌合体蛋白可以成为药物输送到大脑的有用工具的假设。然而，当大鼠通过i.p.增加注射TAT-HuIFN-β时，我们未能证明HuIFN-β进入大脑的传递增强。当注射药物的浓度改变时，结果也是一样的。我们的结论是，HuIFN-β不能穿透血脑屏障并发挥足够的抗肿瘤作用来治疗恶性脑肿瘤

TAT chimera proteins have the potential as a carrier for delivering proteins through the blood-brain barrier and enable the target substance to reach the brain parenchyma by systemic administration. We have investigated the possibility of using blood-brain barrier (BBB) permeable TAT chimera Interferon-β for the treatment of malignant gliomas. We have performed an RT-PCR of human Interferon-β (HuIFN-β) with His-tag and TAT-protein transduction domain (PTD) attached on the C-terminus. The DNA sequence was confirmed and subcloned into a plasmid expression vector. The designed plasmid was transformed into E.coli and large scale protein expression was performed. TAT-HuIFN-β was extracted as insoluble fraction and solublized by SB3-l4. TAT-HuIFN-β was purified by Ni matrix, with protein refolding. As a control study, TAT-HA peptide was injected into a rat by i.p. and high concentration of HA antigen was detected in the rat brain compared to injection of HA alone. This result supports the hypothesis that TAT chimera proteins can be a useful tool of drug delivery into the brain. However, when TAT-HuIFN-β was injected into rats by i.p. increased we failed to demonstrate the enhanced delivery of HuIFN-β into the brain. This was also the same when the concentration of the injected drug was changed. We conclude that HuIFN-β cannot penetrate the BBB and exert an enough anti-tumoral effect for the treatment of malignant brain tumors