TAT蛋白介导生物活性蛋白到达缺血灶的研究

Study on TAT protein mediate delivery of biologically active protein to ischemic regions

目的 :以 β 半乳糖苷酶 (β Gal)为目的蛋白 ,观察TAT蛋白介导其到达缺血灶的情况、及有无血脑屏障 (BBB)的破坏。方法 :构建TAT β Gal融合基因表达质粒 ,转染大肠杆菌BL2 1(DE3)。利用鏊合金属亲合层析法纯化蛋白。将 36只SD大鼠随机分为 3组。其中 2组 ,采用经颈外动脉血管内直接线栓闭塞法 ,建立可复流的大脑中动脉闭塞(MCAO )模型 ,分别腹腔注射TAT β Gal 5mg·kg- 1,β Gal 5mg·kg- 1,并同时注射Evan’s蓝。另一组为假手术组 ,腹腔注射TAT β Gal 5mg·kg- 1。3组分别于给药后 30min ,2h ,4h断头取脑、快速冰冻切片 ,脑切片间隔行x Gal及TTC染色。结果 :MCAO模型SD大鼠 ,腹腔注射TAT β Gal融合蛋白后 ,30min时脑组织各个区域及缺血灶区即可检测到外源性 β Gal的活性 ,4h达高峰 ,同时BBB没有被破坏。而对照组 ,脑组织各个区域未检测到外源性β Gal的活性。结论 :TAT蛋白能有效地介导生物活性蛋白通过BBB进入缺血灶区在脑组织内达到有效浓度。

AIM: To observe if TAT protein can mediately deliver other protein to ischemic regions and have any damage to blood brain barrier. METHODS: Firstly, a bacteria expression vector, pET28a TAT LacZ was transformed into bacteria E.coli BL21(DE3). The fusion protein could be induced by 0.5 1 mmol·L -1 IPTG and was purified by Chelating Sepharose Fast Flow. Thirty six Sprarue Danley(SD) rats were randomly divided into three groups, two opera, one sham opera. Middle cerebral artery occlusion(MACO) was achieved using an nylon suture embolization and reperfusion was performed by the suture withdrawal. In two opera groups TAT β Gal and control β Gal 5 mg·kg -1 was intraperitoneally injected into two MACO groups rats, respectively. TAT β Gal 5 mg·kg -1 was intraperitoneally injected into sham opera group. Then TTC and X Gal stained brain section from each group rats in various time. RESULTS: β Gal activity can be detected in each brain areas and ischemic regions 30 min after ip injection with TAT β Gal, by 4 h all region of the brain showed strong β Gal activity. In brain section from rat control group β Gal showed no β Gal activity. CONCLUSION: The TAT protein can mediately deliver biologically active protein to ischemic regions efficiently.