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500 trg 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各组均町观察到绿色荧光,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.
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-l-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 after0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h. Transduction efficiency was evaluated by itnmunofluorescence 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-1-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-1-CAT can be transduced into rat hearts in vivo in a naturally active form in a time- dependent manner, which provides an experimental basis of PEP-1-CAT for the prevention and therapy of myocardial ischemia-reperfusion injury.
PEP-1Catalasemyocardial reperfusion injurytransductionin vivo
NETL
NSTL
万方数据
维普
