DN—JNK基因重组腺病毒的构建和鉴定  

Construction and identification of dominant-negative c-Jun N-terminal kinase （DN-JNK） recombinant adenovirus

作　　者：张佳妮[1] 刘慧霞[1] 陈金虎[1] 郭敏[1] 全养雅[1] 谭莺[1] 

机构地区：[1]中南大学湘雅医院老年病科,湖南长沙410008

出　　处：《中国医师杂志》2009年第1期28-31,共4页Journal of Chinese Physician

基　　金：国家自然科学基金资助项目（30670945）;国家人事部留学人员科技活动项目择优资助经费项目（2006-164）;湖南省自然科学基金资助项目（07JJ5108）

摘　　要：目的制备表达人c-jun氨基末端激酶（JNK）复制缺陷型重组腺病毒（Ad—DN—JNK），并通过动物实验进行功能鉴定。方法将重组穿梭载体pAdTrack—CMV—DN—JNK线性化后，与pAdEasy—1共转化大肠杆菌BJ5138，进行同源重组得到重组腺病毒载体。将重组腺病毒载体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒，并经PCR及DNA测序鉴定。Western blot检测Ad—DN-JNK在SD大鼠肝组织中JNK1蛋白表达情况，及胰岛素受体底物1丝氨酸307（IRS-1^Ser307）磷酸化水平。结果JNK重组腺病毒载体能有效转染HEK293细胞并在细胞内成功包装，5d后观察到绿色荧光蛋白（GFP）明显表达，搜集的病毒经过PCR扩增得到了特定JNK基因片段并测序鉴定。所制备的Ad—DN—JNK滴度为2．5×10^10pfu／ml，动物实验证实构建的Ad—DN-JNK能有效在肝组织表达。结论该研究成功构建了DN—JNK重组腺病毒载体及相应重组腺病毒颗粒，动物实验证明Ad—DN—JNK能有效介导DN—JNK基因蛋白表达并下调IRS-1^Ser307磷酸化水平。为进一步研究JNK的作用及应用DN—JNK进行相关疾病的基因治疗奠定基础。Objective To construct and identify replication deficient recombinant adenovirus expressing human c-Jun N-terminal kinase（JNK） by homologous recombination adenovirus dominant-negative type JNK（Ad-DN-JNK）. Methods The linearized recombinant shuttle vector pAdTrack-CMV-DN-JNK was co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for recombinant adenoviral vector. The recombinant adenoviral vector was transfeeted into HEK293 packing cells to construct replication deficient recombinant adenovirus, and then the recombinant adenovirus was detected by PCR and DNA sequencing. Western blot analysis was utilized to detect the expression of Ad-DN-JNK and the level of insulin receptor substrate 1 Serine307 phosphorylation. Results JNK recombinant adenoviral vector could be effectively transfeeted into HEK 293 cells and successfully packed by intracellular enzyme. The expression of green fluorescent protein （GFP） was observed on the 5th day after transfeetion. The fragment of JNK gene was amplified by PCR and identified by sequencing. The titer of the prepared Ad-DN-JNK is 2. 5 × 10^10 pfu/ml. The animal experiment confirmed that constructed Ad-DN-JNK could be effectively expressed in liver tissue. Conclusion The research successfully constructed recombinant adenoviral vector and recombinant adenoviral particle. Animal experiment demonstrated the Ad-DN-JNK could effectively mediated the expression of DN-JNK gene and down-regulated the level of IRS1Serine307 phosphorylation. The achievement laid a foundation for further investigation of the function and application of JNK.

关 键 词：原癌基因蛋白质c-jun 重组蛋白质类 腺病毒科 

分 类 号：Q[生物学]