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  

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

作　　者：张永军[1,2] 王家宁[1,2] 唐俊明[1] 杨建业[1] 郭凌郧[1] 黄永章[1] 

机构地区：[1]郧阳医学院附属人民医院临床医学研究所,湖北十堰442000 [2]武汉大学人民医院心内科,湖北武汉430060

出　　处：《第四军医大学学报》2009年第3期225-228,共4页Journal of the Fourth Military Medical University

基　　金：湖北省高等学校优秀中青年科技创新团队(T200811)

摘　　要：目的:观察PEP-1介导CAT转导入心肌组织的能力,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0.5,1,2,4,8,24 h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8 h时强度最大,同时Western Blot也检测到了目的蛋白,8 h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8 h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.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.

关 键 词：PEP-1 过氧化氢酶 心肌再灌注损伤 转导 在体 

分 类 号：Q784[生物学—分子生物学]