心肌肌钙蛋白T启动子经两步转录扩增系统提高目的基因表达的腺相关病毒载体构建

Construction of adeno-associated virus vector for improving expression of target gene by two-step transcription amplification system with myocardial specific promoter

目的构建心肌肌钙蛋白T(cardiac troponin T, cTnT)启动子经两步转录扩增(two-step transcription amplification, TSTA)系统调控下增强绿色荧光蛋白(enhanced green fluorescent protein, EGFP)作为目的基因表达荧光标签的重组腺相关病毒(recombinant adeno-associated virus, rAVV)载体(pAAV-cTnT-TSTA-EGFP),验证cTnT启动子经TSTA系统调控后的转录效果,并包装、纯化相应的腺相关病毒(adeno-associated virus, AVV)。方法利用同源重组法构建载体质粒pAAV-cTnT-TSTA-EGFP,经基因测序鉴定其序列正确性。将对数生长期人源胚胎肾细胞HEK-293T分为cTnT组(转染质粒pAAV-cTnT-EGFP),TSTA组(转染质粒pAAV-cTnT-TSTA-EGFP),巨细胞病毒(cytomegalovirus, CMV)组(转染质粒pAAV-CMV-EGFP),对照组(不转染质粒)。转染72 h,倒置荧光显微镜下观察4组EGFP荧光表达情况,应用流式细胞仪检测4组EGFP表达量。使用pAAV-cTnT-TSTA-EGFP、pAAV-R2C9及pHelper辅助质粒共转染HEK-293T细胞进行rAVV的包装,72 h后收集细胞并进行rAVV纯化,采用Western blot法检测rAVV包装是否成功,采用实时荧光定量PCR法测定纯化后病毒的滴度。结果转染72 h, cTnT组EGFP荧光表达少,TSTA组EGFP荧光表达与CMV组基本相同;流式细胞仪检测结果显示,TSTA组EGFP表达量至少较cTnT组增强50倍,与CMV组仅相差3倍;Western blot检测结果显示,纯化后rAVV包装成功,纯化后的rAAV-cTnT-TSTA-EGFP溶液病毒滴度为2.74×10;GC/mL。结论 TSTA系统可明显增加心肌特异性cTnT启动子对目的基因的转录表达水平。

Objective To construct recombinant adeno-associated virus(rAAV) vector expressed by enhanced green fluorescent protein(EGFP) as target genes by two-step transcription amplification of cardiac troponin T(cTnT) promoter, and to verify the enhanced effect of TSTA system, package and purify the corresponding adeno-associated virus(AAV). Methods The rAAV vector plasmid pAAV-cTnT-TSTA-EGFP was constructed by homologous recombination, and its sequence was verified by sequencing. The HEK-293 T cells in logarithmic growth phase were divided into cTnT group(transfected with plasmid pAAV-cTnT-EGFP), TSTA group(transfected with plasmid pAAV-cTnT-TSTA-EGFP), cytomegalovirus(CMV) group(transfected with plasmid pAAV-CMV-EGFP), and negative control group(transfected with no plasmid). After 72 h of transfection, the green fluorescent protein expression of EGFP in each group was detected by inverted fluorescence microscope, and the relative expression of EGFP was detected by flow cytometry. HEK-293 T cells were co-transfected with three plasmids for packaging of AAV, and the viruses were collected and purified 72 h later. After purification, Western blot was used to detect whether the virus package was successful. The titer of purified virus was determined by real-time fluorescence quantitative PCR. Results The fluorescent expression of EGFP was less in cTnT group,and was similar in TSTA group compared with CMV group.The GFP expression was about 50 times higher in TSTA group than that in cTnT group,and 3 times less than CMV group.The rAAV was successfully packaged after purification.The titer of purified rAAV-cTnT-TSTA-EGFP virus was 2.74×10;GC/mL.Conclusion The TSTA system significantly enhances the transcription of target gene by cTnT promoter.