摘要
目的构建含大鼠降钙素相关基因肽(CGRP)基因的慢病毒表达载体,为后续转染目的细胞并研究CGRP的功能奠定基础。方法通过基因工程技术将CGRP基因克隆到穿梭质粒中,构建Puc57-CGRP质粒,用双酶切法构建慢病毒表达载体(pLenO-DCE-CGRP),将该质粒载体与4种辅助包装质粒共转染293T细胞,转染的293T细胞继续培养48h后,收集其上清液,浓缩得到高滴度的慢病毒液,然后采用倍比稀释法和流式细胞术检测病毒滴度,并通过实时定量聚合酶链反应(PCR)检测293T细胞中CGRP基因的表达。结果成功构建了pLenO-DCE-CGRP的重组慢病毒载体,滴度为5.1×108TU/mL。结论成功构建了含CGRP基因高滴度的慢病毒载体,为后续转染间充质干细胞(MSC)并研究CGRP的功能奠定了基础。
Objective To construct lentiviral vector carrying rat＇s calcitonin gene-related peptide（CGRP） gene for the following- up study on the function of CGRP. Methods CGRP gene segment was subcloned into shuttle plasmid,become Pue57-CGRP. The pLenO-DCE-CGRP expression vector was be constructed by double digests. The pLenO-DCE-CGRP and 4 auxiliary packaging plasraids were co-transfected into 293T cells. Cells were cultured for 48 hours. The supernant was collected and concentrated,and then the viral titers were tested by multiple proportions dilution method and flow cytometer. The expression levels of CGRP were detected in CGRP-modified 293T cells by Real-time PCR. Results The results of digestion and sequencing show that the pLenO-DCE- CGRP vector was constructed successfully. The titer of the lentiviral particles was 5.1 × 10^8 TU/mL. Conclusion The high-titer lentvirus vector containing CGRP gene is constructed successfully, which lay a foundation for transfecting mesenchymal stem cell （MSC） and studying the function of CGRP.
出处
《重庆医学》
CAS
CSCD
北大核心
2013年第34期4157-4159,共3页
Chongqing medicine
基金
国家自然科学基金资助项目(81060014)
作者简介
陈攀科（1982-），硕士，主治医师，主要从事血管及冠心病介入的研究。
通讯作者，Tel：15085117532；E-mail：Shibei2147@163．com。
