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目的:原核表达、纯化PTD-XIAP融合蛋白,鉴定其血脑屏障的通透功能。方法:反转录大鼠脑组织RNA,用PCR法扩增编码XIAP蛋白BIR1-2、BIR3-RING及BIR-2的cDNA片断,重组入pTrans Vector表达质粒并转化大肠埃希菌BL21;以IPTG诱导表达,用NTA-Ni层析柱分离纯化,Western blot鉴定。PTD-BIR3-RING经腹腔注入小鼠体内,用免疫荧光法检测脑组织中的分布。结果:PTDBIR3-RING得到表达,相对分子质量约30×10~3,与6×His单克隆抗体有特异性反应。注入小鼠体内后,脑组织中广泛分布有阳性细胞。结论:重组PTD-BIR3-RING具有良好的血脑屏障穿透功能。
OBJECTIVE: To purify PTD-XIAP fusion protein by prokaryotic expression and identify its permeability function of blood-brain barrier. Methods: RNA of rat brain tissues was reverse transcribed. CDNA fragments encoding BIR1-2, BIR3-RING and BIR-2 of XIAP were amplified by PCR. Recombinant pTrans Vector was transformed into E. coli BL21. Induce the expression, using NTA-Ni column separation and purification, Western blot identification. PTD-BIR3-RING was intraperitoneally injected into mice and the distribution of brain tissue was detected by immunofluorescence. Results: PTDBIR3-RING was expressed with relative molecular mass of about 30 × 10-3, which reacted specifically with 6 × His monoclonal antibody. After injection into the body of a mouse, positive cells are widely distributed in the brain tissue. Conclusion: Recombinant PTD-BIR3-RING has a good blood-brain barrier penetrating function.