细胞穿透肽PEP-1介导人过氧化氢酶转导入在体大鼠心肌组织

目的：观察PEP-1介导CAT转导人心肌组织的能力，检测转导的CAT是否具有天然活性．方法：用基因工程手段表达并纯化出PEP-1-CAT和CAT融合蛋白．以生理盐水为对照，分别从SD大鼠尾静脉注射500μg PEP-1-CAT和CAT融合蛋白，在0．5，1，2，4，8，24h时将其处死，取其心脏，免疫荧光及Western Blot检测CAT的转导能力，用试剂盒检测心肌CAT的酶活性．结果：SDS—PAGE和Western Blot证实目的蛋白PEP-1-CAT和CAT得到了高表达，且纯度在90％左右．生理盐水组及CAT组，荧光显微镜下均未见到绿色荧光，Western Blot亦未检测到目的蛋白；PEP-1-CAT各组均可观察到绿色荧光，且8h时强度最大，同时Western Blot也检测到了目的蛋白，8h时量最多；酶活性检测显示，CAT组与生理盐水对照组差别无统计学意义，而PEP-1-CAT组随着时间的延长，心肌组织中CAT的活性逐渐增加，在8h时达到峰值，为对照组的4．20倍．结论：PEP-1能够介导CAT以天然活性方式转导入大鼠心肌组织，且其转导呈时间依赖性．

Transduction of catalase into rat hearts in vivo mediated by cell-penetrating peptide PEP-1

AIM： To investigate the transduction efficiency of purified PEP-1-CAT fusion protein and to evaluate its enzyme activity. METHODS： The constructed pET15b-PEP-1-CAT and pET15b-CAT were transformed into E. coli BL21 （ DE3 ） and the protein expression was induced by IPTG. PEP-1-CAT and CAT （500 μg） were injected into SD rats via caudal vein, respectively and normal saline （NS） was used as the control. The hearts were harvested after 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h. Transduction efficiency was evaluated by immunofluoreseence technique and Western Blot. The CAT activity was measured according to the manufacturer＇s protocols. RESULTS： It was confirmed by SDS- PAGE and Western Blot that the target protein of PEP-1-CAT and CAT was expressed efficiently and the purity was about 90%. No green fluorescence was observed in NS group and CAT group and no target protein was detected by Western Blot in CAT group. Bright green fluorescence was observed in PEP-I-CAT groups at different time points and the 8 h group was the brightest. The target protein was detected by Western Blot in PEP-1-CAT group and the quantity of 8 h group was the brightest. No statistical difference in CAT activity was found between CAT groups and NS group （P 〉 0.05 ）, and with the lapse of time, the CAT activity of the transduced PEP-1-CAT increased gradually. The activity was the highest in 8h group and the activity of 8 h group was 4.2 times that of the NS group. CONCLUSION： PEP-l-CAT can be transduced into rat hearts in vivo in a naturally active form in a timedependent manner, which provides an experimental basis of PEP- 1-CAT for the prevention and therapy of myocardial ischemia reperfusion injury.