小鼠降钙素基因相关肽基因重组腺病毒载体的构建及其在心肌细胞的表达  

Construction of recombinant adenovirus vector of calcitonin gene-related peptide gene and transfection to neonatal rat cardiomyocytes

作　　者：孙智慧[1] 韩杰[1] 邵蕾[1] 王利宏[1] 宋俊贤[1] 魏钟海[1] 郑良荣[1] 

机构地区：[1]浙江大学医学院附属第一医院心血管内科,浙江杭州310003

出　　处：《浙江大学学报（医学版）》2010年第3期311-317,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：浙江省科技攻关计划(2006C33010);浙江省部共建计划(wkj2008-2-019)

摘　　要：目的:利用AdEasy腺病毒载体系统,构建小鼠降钙素基因相关肽(calcitonin gene-related peptide,CGRP)基因重组腺病毒载体,并验证其在心肌细胞中的表达。方法:采用RT-PCR方法扩增出含有小鼠CGRP全长cDNA的基因片段,并将其插入PUC57载体,经EcoRⅤ和SalⅠ双酶切,将小鼠CGRP基因亚克隆至穿梭质粒pShuttle-CMV,形成重组质粒pShuttle-CMV-CGRP。经Pm eⅠ酶切线性化后,在大肠杆菌BJ5183中与腺病毒骨架质粒pAdEasy-1同源重组产生重组腺病毒质粒AdEasy-pShuttle-CGRP。重组腺病毒质粒在XL-Gold细胞中扩增,经PacⅠ酶切线性化后,转染人胚肾293细胞进行重组腺病毒的包装,并经氯化铯纯化得到重组腺病毒AdEasy-pShuttle-CGRP。用纯化后的重组腺病毒转染原代培养的乳鼠心肌细胞,荧光显微镜下观察转染效果。通过尾静脉导入重组腺病毒7 d后,放免法测定心肌中CGRP的表达量。结果:测序证实PUC57-CGRP重组质粒中含有小鼠CGRP基因的cDNA;重组穿梭质粒pShuttle-CMV-CGRP经双酶切鉴定后可见约7.5 kb和423 bp片段;重组腺病毒质粒AdEasy-pShuttle-CGRP经PacⅠ酶切可见长约3kb与30 kb DNA片断。转染293细胞进行包装,7 d后细胞出现病变效应,回收病毒重复感染293细胞,在荧光显微镜下可见绿色荧光蛋白表达。提取病变293细胞中重组腺病毒进行目的基因的PCR鉴定为阳性。构建好的重组腺病毒经纯化后具有很高的滴度,并且成功转染了原代培养的乳鼠心肌细胞。通过放免法证实,该载体可以显著增加小鼠心脏中CGRP的表达。结论:成功构建携带CGRP基因的重组腺病毒载体,该重组腺病毒载体AV-CGRP可以显著增加心脏中CGRP的表达量,为进一步研究CGRP基因治疗奠定了基础。Objective： To construct a recombinant adenovirus vector of calcitonin gene-related peptide（CGRP） by AdEasy system and to validate its expression in myocardial cells.Methods： The full-length of CGRP gene cDNA was acquired by RT-PCR and cloned into pShuttle-CMV.After linearization with Pme Ⅰ,the recombinant plasmid（pShuttle-CMV-CGRP） was transformed into E.coli BJ5183 by electroporation to construct the recombinant adenovirus plasmid AdEasy-pShuttle-CGRP.The recombinant adenovirus plasmids were transformed into E.coli XL10-Gold cells to be amplified.Then the recombinant plasmid was digested with Pac Ⅰ and transfected to 293 cells to package recombinant adenovirus particles.PCR technique was used to detect target gene.The recombinant adenovirus particles were purified by CsC1 density gradient.The purified recombinant adenovirus was transfected to neonatal rat cardiomyocytes,and the recombinant adenovirus production was observed by fluorescent microscope.Expression of CGRP in hearts 7 days after intravenous delivery of advenoviral vectors AV-CGRP was determined by radioimmunoassay.Results： The RT-PCR products confirmed a full-length cDNA of CGRP gene in PUC57 by sequencing.The corresponding double endonuclease and PCR analysis certified the successful cloning of the gene into the pShuttle-CMV.The recombinant adenovirus plasmid AdEasy-pShuttle-CGRP was digested by Pac Ⅰ endonuclease to form the typical DNA segments,whose length was about 3 kb and 30 kb.PCR analysis and fluorescent microscope observation confirmed that the CGRP gene was inserted into the adenovirus vector with very strong power of transfection.The recombinant adenovirus particles infected neonatal rat cardiomyocytes successfully.Radioimmunoassay showed that delivery of AV-CGRP significantly increased the expression of CGRP in mice hearts.Conclusions： The recombinant adenovirus vector of CGRP gene has been constructed,and it can infect neonatal rat cardiomyocytes successfully.Somatic delivery of CGRP gene can significantly increase

关 键 词：降钙素基因相关肽 重组 遗传 腺病毒科/遗传学 遗传载体 基因表达 基因疗法 心肌/细胞学 

分 类 号：Q78[生物学—分子生物学]