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。方法将重组穿梭载体pAdTrack-CMV-WT-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。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后可以观察到绿色荧光蛋白（GFP）明显表达，搜集的病毒经过PCR扩增得到特定JNK基因片段并测序鉴定，动物实验证实构建的Ad-WT-JNK能有效在肝组织表达。结论该研究成功构建了JNK重组腺病毒载体及相应重组腺病毒颗粒，为进一步研究JNK的作用及应用JNK进行相关疾病的基因治疗奠定了基础。
Objective To construct replication deficient recombinant adenovirus expressing human c-Jun N-terminal kinase by homologous recombination. Methods The linearized recombinant shuttle vector pAdTrack-CMV-WT-JNK was co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for recombinant adenoviral vector, The recombinant adenoviral vector was transfected into HEK293 packing cells to construct replication deficient recombinant adenovirus, and then the recombinant adenovirus was detected by PCR and DNA sequencing. Results JNK recombinant adenoviral vector was effectively transfected into HEK 293 cells and was successfully packed by intracellular enzyme. The expression of green fluorescent protein （GFP） was observed on the 5th day after transfection. The fragment of JNK gene was amplified by PCR and identified by sequencing. The animal experiment confirmed that Ad-WT-JNK was effectively expressed in liv- er tissue, Conclusion The research successfully constructed recombinant adenoviral vector and recombinant adenoviral particle. And the achievement laid a foundation for further investigation of the function and application of JNK.
出处
《中国医师杂志》
CAS
2008年第3期309-311,共3页
Journal of Chinese Physician
基金
基金项目：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）
湖南省科技厅资助项目（06SK3020）
湖南省卫生厅科研基金项目（2006-045）.
