CAJ | 학술논문

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pAdTraek-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.将重组腺病毒栽体转染入包装细胞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-1Ser307)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,5 d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2.5×1010pfu/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-1Ser307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.
목적 제비표체인c-jun안기말단격매(JNK)복제결함형중조선병독(Ad-DN-JNK).병통과동물실험진행공능감정.방법 장중조천사재체pAdTraek-CMV-DN-JNK선성화후,여pAdEasy-1공전화대장간균BJ5138,진행동원중조득도중조선병독재체.장중조선병독재체전염입포장세포HEK293내제비복제결함형중조선병독,병경PCR급DNA측서감정.Western blot검측Ad-DN-JNK재SD대서간조직중JNK1단백표체정황,급이도소수체저물1사안산307(IRS-1Ser307)린산화수평.결과 JNK중조선병독재체능유효전염HEK293세포병재세포내성공포장,5 d후관찰도록색형광단백(GFP)명현표체,수집적병독경과PCR확증득도료특정JNK기인편단병측서감정.소제비적Ad-DN-JNK적도위2.5×1010pfu/ml,동물실험증실구건적Ad-DN-JNK능유효재간조직표체.결론 해연구성공구건료DN-JNK중조선병독재체급상응중조선병독과립,동물실험증명Ad-DN-JNK능유효개도DN-JNK기인단백표체병하조IRS-1Ser307린산화수평.위진일보연구JNK적작용급응용DN-JNK진행상관질병적기인치료전정기출.
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-l 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 edenovirns WaS detected by PCR and DNA sequencing.Western blot analysis was utilized to detect the Cxpression of Ad-DN-JNK and the level of insulin receptor substrate l Serine307 phosphorylation.Results JNK recombinant adenoviral vectorcould be effectively transfeeted into HEK 293 cell and 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 titer of the prepared Ad-DN-JNK is 2.5×1010 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 IRSlscfine307 phosphorylation.The achievement laid a foundation for further investigation of the function and application of JNK.