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*★
杨丽华1，张学光1，王如兴2，陈永井1，吕海军1，张 燕1，李肖蓉2
课题背景：成功应用成纤维细胞生长因子2基因治疗的技术关键步骤是成纤维细胞生长因子2基因的克隆及其真核表达载体的构建。国内外数据库中关于大鼠成纤维细胞生长因子2分子的克隆和及其真核表达载体的构建的研究很少。
应用要点：在克隆和扩增大鼠成纤维细胞生长因子的过程中，由于此基因本身结构的特异性，无法扩增出与Genebank中百分之百吻合的基因片段，最终扩增出的基因序列有一个无意突变，不影响其在体内的表达。
同行评价：从文章叙述看，研究者有较多自己的经验，应该有过多次的探索试验，为文章最后的结果奠定了基础。具有一定的新颖性。从研究者描述的操作方法看，其过程正确，结果应该能够重复，实验过程及结果对其他研究者均具有借鉴作用。
摘要
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。
关键词：成纤维细胞生长因子2；心肌/细胞学；载体构建；大鼠；组织构建
杨丽华，张学光，王如兴，陈永井，吕海军，张燕，李肖蓉. 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 [J].中国组织工程研究与临床康复，2008，12(15):2875-2878 [www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2875(ps).pdf]
1苏州大学医学生物技术研究所江苏省干细胞重点实验室，江苏省苏州市 215007； 2无锡市第一人民医院心内科，江苏省无锡市
214002
杨丽华★，女，1981年生，陕西省宝鸡市人，汉族，苏州大学在读硕士，主要从事干细胞与临床、分子免疫方向研究。
lihuayang1981@
yahoo.com.cn
通讯作者：李肖蓉，硕士生导师，教授，无锡市第一人民医院心内科，江苏省无锡市
214002 lixiaorong.dr@163.
com
江苏省卫生厅医学重点实验室开放课题资助项目（WK0512）*
中图分类号:R329.114
文献标识码:B
文章编号:1673-8225
(2008)15-02875-04
收稿日期：2007-09-05
修回日期：2008-01-15
(07-50-9-4837/W·Y)
Cloning of fibroblast growth factor-2 and construction of its eukaryotic cell expressing vector
Abstract
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 carried out in the Biotechnology Institute of Soochow University from March to June in 2007.①Materials: 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.②Experimental 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 bglⅡ and pst Ⅰ, the cut gene fragments and PIRES2-EGFP were ligated with T4DNA ligase to construct eukaryotic expressing vector.
RESULTS: ①Neonatal SD rat ventricular cells with typical morphology were cultured successfully.②The rat FGF-2 DNA sequence obtained in this experiment was consistent with that included in Genebank.③The 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.
Yang LH, Zhang XG, Wang RX, Chen YJ, Lü HJ, Zhang Y, Li XR. Cloning of fibroblast growth factor-2 and construction of its eukaryotic cell expressing vector.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(15):2875-2878(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2875(ps).pdf]
1Biotechnology Institute of Soochow University, Stem Cell Key Laboratory of Jiangsu Province, Suzhou 215007, Jiangsu Province, China; 2Department of Cardiology, Wuxi First People's Hospital, Wuxi 214002, Jiangsu Province, China
Yang Li-hua★, Studying for master's degree, Biotechnology Institute of Soochow University, Stem Cell Key Laboratory of Jiangsu Province, Suzhou 215007, Jiangsu Province, China
lihuayang1981@
yahoo.com.cn
Correspondence to: Li Xiao-rong, Tutor of master, Professor, Department of Cardiology, Wuxi First People's Hospital, Wuxi 214002, Jiangsu Province, China
lixiaorong.dr@
163.com
Supported by: Open Program of Medical Key Laboratory by Jiangsu Province Health Bureau, No. WK0512*
Received: 2007-09-05
Accepted: 2008-01-15
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