Sun Zhi-hui, Han Jie, Shao Lei, Wang Li-hong, Song Jun-xian, Wei Zhong-hai, Zheng Liang-rong
Department of Cardiology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2010 May;39(3):311-7. doi: 10.3785/j.issn.1008-9292.2010.03.016.
To construct a recombinant adenovirus vector of calcitonin gene-related peptide (CGRP) by AdEasy system and to validate its expression in myocardial cells.
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 adenoviral vectors AV-CGRP was determined by radioimmunoassay.
The RT-PCR products confirmed a full-length cDNA of CGRP gene in PUC(57) 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.
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 the expression of CGRP in mice hearts. The results may provide a sound foundation for further study on the value of CGRP as the target for gene therapy in both laboratory and clinical trials.
利用AdEasy系统构建降钙素基因相关肽（CGRP）重组腺病毒载体，并验证其在心肌细胞中的表达。
通过RT-PCR获得CGRP基因cDNA全长，并克隆至pShuttle-CMV。经Pme I线性化后，将重组质粒（pShuttle-CMV-CGRP）通过电穿孔法转化至大肠杆菌BJ5183，构建重组腺病毒质粒AdEasy-pShuttle-CGRP。将重组腺病毒质粒转化至大肠杆菌XL10-Gold细胞进行扩增。然后用Pac I酶切重组质粒并转染至293细胞以包装重组腺病毒颗粒。采用PCR技术检测靶基因。通过CsC1密度梯度法纯化重组腺病毒颗粒。将纯化后的重组腺病毒转染至新生大鼠心肌细胞，通过荧光显微镜观察重组腺病毒的产生情况。采用放射免疫分析法测定腺病毒载体AV-CGRP静脉注射7天后小鼠心脏中CGRP的表达。
RT-PCR产物经测序证实PUC(57)中存在CGRP基因的全长cDNA。相应的双酶切和PCR分析证实该基因成功克隆至pShuttle-CMV。重组腺病毒质粒AdEasy-pShuttle-CGRP经Pac I内切酶酶切形成典型的DNA片段，长度约为3 kb和30 kb。PCR分析和荧光显微镜观察证实CGRP基因已插入腺病毒载体，且转染能力很强。重组腺病毒颗粒成功感染新生大鼠心肌细胞。放射免疫分析表明，AV-CGRP的递送显著增加了小鼠心脏中CGRP的表达。
已构建CGRP基因重组腺病毒载体，且能成功感染新生大鼠心肌细胞。CGRP基因的体细胞递送可显著增加小鼠心脏中CGRP的表达。这些结果可为进一步研究CGRP作为基因治疗靶点在实验室和临床试验中的价值提供良好基础。
