中文摘要：

目的：构建SD大鼠心肌肌钙蛋白Ⅰ（cardiac troponinⅠ,cTnⅠ）天冬氨酸（Asp,D）128酪氨酸（Tyr,Y）突变（cTnⅠD128Y）重组腺病毒载体,并鉴定。方法：构建野生型SD大鼠cTnⅠ基因全长ORF区克隆,在此基础上,采用PCR定点突变方法对cTnⅠ第128位氨基酸位点引入D128Y突变,将野生型和突变型cTnⅠ基因亚克隆至Adeno-X腺病毒载体,转染HEK 293细胞,包装形成野生型和突变型（Ad-cTnⅠD128Y）重组腺病毒载体;并采用DNA测序、免疫印迹和细胞培养等方法进行鉴定。结果：PCR及测序结果表明,重组腺病毒克隆载体序列正确,免疫印迹证明有融合突变蛋白表达,该突变病毒转染心肌细胞见绿色荧光蛋白表达。结论：本研究成功构建了大鼠cTnⅠD128Y突变重组腺病毒载体,为探讨该突变致心肌肥大的胞内信号通路研究提供了基础。

英文摘要：

Objective： To construct and assess a recombinant adenovirus expression vector of rat’s cardiac troponin Ⅰ Asp128Tyr gene mutation（cTnⅠ D128Y）.Methods： On the basis of ORF clone construction of cardiac troponin I gene of SD rat,the cTnⅠ D128Y mutation was induced by PCR based site-directed mutagenesis.With EGFP as a reporter gene,the coding region of mutant cTnⅠ gene were subcloned into Adeno-X viral DNA to form a recombinant adenoviral plasmid,which were packed into infectious recombinant adenoviral particles（cTnⅠ D128Y GFP） by transfecting HEK293 cells.Results： The DNA sequence of Ad cTnⅠ D128Y was proved to be correct by PCR and sequencing,and its protein expression was identified by Western Blot analysis.Furthermore,GFP could be seen in cardiac cells which infected with the recombinant adenovirus expression vector.Conclusion： The successfully constructed recombinant adenovirus Ad cTnⅠ D128Y has provided basis for further researches on its functional characteristics and mechanisms.