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

达特嵌合体蛋白具有作为通过血脑屏障递送蛋白质的载体的潜力，并使靶物质能够通过全身给药到达脑实质。我们研究了使用血脑屏障（BBB）可渗透达特嵌合体干扰素-β治疗恶性胶质瘤的可能性。我们对人β干扰素（HuIFN-β）进行了RT-PCR，其C-末端带有His标签和TAT蛋白转导结构域（PTD）。确认DNA序列并亚克隆到质粒表达载体中。将设计的质粒转化到大肠杆菌中并进行大规模蛋白表达。将TAT-HuIFN-β作为不溶性级分提取并通过SB 3 - 14溶解。采用Ni基质纯化TAT-HuIFN-β，并进行蛋白复性。作为对照研究，将TAT-HA肽通过腹膜内注射到大鼠中，与单独注射HA相比，在大鼠脑中检测到高浓度的HA抗原。这一结果支持了达特嵌合体蛋白可以是将药物递送到脑中的有用工具的假设。然而，当通过腹膜内增加将TAT-HuIFN-β注射到大鼠中时，我们未能证明HuIFN-β向脑中的递送增强。当注射药物的浓度改变时，这也是相同的。结论：HuIFN-β不能穿透血脑屏障，不能发挥足够的抗肿瘤作用

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