大鼠成纤维细胞生长因子2分子克隆及其真核表达载体的构建

Cloning of fibroblast growth factor-2 and construction of its eukaryotic cell expressing vector

目的:实验旨在克隆大鼠成纤维细胞生长因子2CDS区基因全长,并将其于真核表达载体PIRES2-EGFP相连接,构建成纤维细胞生长因子2的真核表达载体。方法:实验于2007-03/06在苏州大学生物技术研究所完成。①实验材料:克隆载体PMD18-T-vector(takara);真核表达载体PIRES2-EGFP由苏州大学第一附属医院消化科叶建新博士惠赠;大肠杆菌TOP10购自英俊公司;清洁级新生一两天SD大鼠2只。②实验过程及评估:体外分离培养新生SD大鼠心室肌细胞。Trizol法抽提心室肌细胞总mRNA,采用反转录-聚合酶链反应的方法反转录获得总的cDNA,聚合酶链反应的方法扩增成纤维细胞生长因子2基因的CDS区片断全长。将扩增出来的基因片断和克隆载体PMD18-T-vector连接,转化TOP10感受态细菌,阳性克隆经电泳,质粒聚合酶链反应和酶切等鉴定正确后送去测序确认。测序正确后抽重组质粒,bglⅡ和pstⅠ同时双酶切PMD18-T-FGF-2和PIRES2-EGFP,将切下的目的基因片段和线性PIRES2-EGFP空质粒载体在T4DNA连接酶作用下进行连接,构建其真核表达载体。结果:①成功培养出了形态典型的SD大鼠的心室肌细胞。②所获的大鼠成纤维细胞生长因子2DNA序列与GeneBank中收录的大鼠成纤维细胞生长因子2序列完全一致。③PIRES2-EGFP-bFGF真核表达载体的构建测序表明,成纤维生长因子2成功插入真核表达载体PIRES2-EGFP之中。结论:实验成功克隆了大鼠成纤维细胞生长因子2基因全长,并构建了其真核表达载体。为转基因细胞制备和移植治疗心肌梗死奠定基础。

AIM： To clone full-length fragments of rat fibroblast growth factor-2 （FGF-2） CDS part, and connect it with PIRES2-EGFP to construct its eukaryotic cell expressing vector. METHODS： The experiment was carded out in the Biotechnology Institute of Soochow University from March to June in 2007.（1） Materials： Cloning vector PMD18-T-vector （takara）; eukaryotic cell expressing vector PIRES2-EGFP was presented by Ye Jian-xin, doctor of Department of Internal Digestion in NO.1 Affiliated Hospital of Soochow University. E. coli top10 was bought from Invitrogen Company; two clearing neonatal SD rats.（2）Experimental protocol and assessment： Neonatal SD rat ventricular muscle cells were cultured in vitro. The total mRNA was extracted from neonatal rat ventricular cells with Trizol method, and reversed transcription-polymerase chain reaction （RT-PCR） was adopted to obtain total cDNA. PCR reaction was used to amplify full-length fragments of FGF-2 gene with cDNA template. Gene fragments were connected with PMD18-T-Vector, and then were transformed into the competence bacteria TOP10, in which positive clones were identified by means of electrophoresis, plasmid PCR and restriction enzyme digestion. Plasmid was recombined after sequencing. PMD18-T-FGF-2 and PIRES2-EGFP were digested with restriction endonuclease bg1Ⅱ and pst Ⅰ, the cut gene fragments and PIRES2-EGFP were ligated with T4DNA ligase to construct eukaryotic expressing vector. RESULTS： （1）Neonatal SD rat ventricular cells with typical morphology were cultured successfully.（2）The rat FGF-2 DNA sequence obtained in this experiment was consistent with that included in Genebank.（3）The sequencing of PIRES2-EGFP-bFGF eukaryotic expressing vector indicated that, FGF-2 had been inserted into PIRES2-EGFP vector. CONCLUSION： The full-length of rat FGF-2 has been successfully cloned, and its eukaryotic expressing vector was constructed. Establishment of FGF-2 expressing vector will be able to lay the foundation for the further research of transgenic cell preparation and transplantation therapy of myocardial infarction.