解偶联蛋白嵌合体及突变体的构建方法  

Construction of chimeras and mutations for uncoupling proteins

作　　者：王超[1] 刘洋[2] 段秀梅[1] 田庄 王银萍[1] 沃夫冈.格莱尔 付彤[4] 

机构地区：[1]吉林大学第一医院病理诊断中心,吉林长春130021 [2]吉林大学第一医院眼科,吉林长春130021 [3]奥地利格拉茨医科大学分子生物学与生物化学研究所 [4]吉林大学第一医院乳腺外科,吉林长春130021

出　　处：《吉林大学学报（医学版）》2013年第1期174-179,共6页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技发展计划项目资助课题(20120733);奥地利格拉茨医科大学博士后基金资助课题(200812014);吉林省科技厅科技发展青年科研基金资助课题(201101054)

摘　　要：目的:构建解偶联蛋白(UCPs)嵌合体和突变体,为阐明解偶联蛋白家族功能结构域的研究提供条件。方法:采用普通PCR和重叠延伸PCR获得UCPs嵌合体基因片段,分别插入pCR2.1测序载体后进行测序,再以限制性内切酶连接方式将嵌合基因定向亚克隆到真核表达质粒pcDNA3.1中,获得pcDNAUCP1UCP2、pcDNAUCP2UCP1和pcDNAUCP3UCP1,以PCR和酶切方法鉴定构建的重组真核表达质粒pcDNA。利用定向点突变产生pcDNA-UCP3R167G/pcDNA-UCP3RE171/172GG,并进行测序证实。结果:PCR和酶切,获得的目的基因与嵌合基因片段中包含的碱基数量吻合;测序结果,UCPs嵌合体基因序列、突变体基因序列与预期设计完全相同。结论:成功地构建了含有UCPs嵌合体和突变体基因重组真核表达质粒。Objective To construct uncoupling proteins （UCPs） chimeras and mutations and provide conditions for study on the functional domains of uncoupling protein family. Methods The gene fragments of UCPs chimera were obtained by routine polymerase chain reaction （PCR） and splicing overlap extension PCR （SOE-PCR）. Then these sequences were respectively cloned into the vector of pCR2.1 and sequenced. The recombinant eukaryotic expression plasmid pcDNA3. I containing UCPs chimera was constructed by restrictive enzyme ligation. The recombinant plasmid of pcDNAUCP1UCP2, pcDNAUCP2ucel and pcDNAUCP3ucvl were identified by PCR and restrictive enzyme digestion. The mutations of pcDNA-UCP3R167G/pcDNA- UCP3RE17/172GG were generated by site- directed mutagenesis and then verified by sequencing. Results The identification of target gene by restrictve enzyme and PCR was the same as expectation. The sequence of chimera gene was completely identical with the expectation device. Conclusion The recombinant eukaryotic expression plasmid containing UCPs chimeras and mutations is constructed successfully.

关 键 词：解偶联蛋白 线粒体 钙离子通道 

分 类 号：Q78[生物学—分子生物学]