Tet-Off诱导的硫氧还蛋白基因重组腺病毒载体的构建  

Construction of recombinant adenovirus vector with TRX gene by Tet-Off induction

作　　者：李艳丽[1] 滕宗艳[1] 张一娜[1] 于卫刚[1] 

机构地区：[1]哈尔滨医科大学附属第二医院老年病科,黑龙江省哈尔滨市150086

出　　处：《实用老年医学》2009年第4期267-269,285,共4页Practical Geriatrics

基　　金：黑龙江省自然科学基金项目(D-200526);哈尔滨市攻关课题资助(2003AA9CS188-5)

摘　　要：目的克隆人硫氧还蛋白(hTRX)基因,并构建含有该目的基因的重组腺病毒载体。方法用内切酶从重组质粒pGEM-TEasy/TRX上切下全长TRX cDNA片段,与穿梭质粒pshuttle2连接,形成pshuttle2-TRX重组质粒,再双酶切pshuttle2-TRX,将带有CMV启动子的目的片段,插入Tet-Off诱导的Adeno-X病毒DNA中,用PCR、双酶切方法进行鉴定。结果对pGEM-T Easy/TRX重组质粒进行序列测定,测定结果与Gene-Bank序列J04026基本一致,其中包括TRX cDNA全长。该实验构建出含TRX的穿梭质粒pshuttle2-TRX,并成功获得Tet-Off诱导的重组腺病毒载体pAdCMV-TRX。结论TRX基因的克隆及其重组腺病毒载体pAdCMV-TRX的成功构建,为进一步研究TRX生物活性及发病与氧化损伤关系密切的疾病治疗提供新的手段,如病毒性心肌炎、动脉硬化、心肌病、糖尿病、Alzheimer's病等。Objective To clone the human thioredoxin gene （hTRX） and to construct the recombinant adenovirus vector containing hTRX. Methods The full length of TRX cDNA, which was obtained from recombinant plasmid pGEM -T Easy/TRX by restriction enzyme digestion, was inserted into pshuttle2 vector to form pshuttle2-TRX recombinant plasmid. Then pshuttle2-TRX was double digested to get the target fragment with CMV promoter which was inserted into DNA of Adeno-X virus induced by Tet-Off. The new plasmid was identified by PCR and restriction enzyme digestion. Results The pGEM -T Easy/TRX recombinant plasmid was sequenced, and the sequence contained the full length of TRXc DNA, which was in accordance with GeneBank sequence J04026. Pshuttle2-TRX shuttle vector containing TRX was constructed. The pAdCMV-TRX recombinant adenovirus vector by Tet-Off induction was gained successfully. Conclusions The successful cloning of TRX gene and construction of its adenovirus vector pAdCMV-TRX provide a new method for further studies concerning the bioactivity of TRX and the diseases closely related to oxidation damage, such as viral myocardi- tis, arteriosclerosis, cardiomyopathy, diabetes mellitus and Alzheimer＇s disease.

关 键 词：硫氧还蛋白 腺病毒 基因重组 

分 类 号：Q784.2[生物学—分子生物学]