Abstract

BACKGROUND:Bone morphogenetic protein-2 (BMP-2) production of targeted cells is promoted by transfection of adenoviral vectors containing gene, but there are some immune responses. Transfection with plasmid as vector holds promise.
OBJECTIVE: To explore the feasibility to construct human bone morphogenetic protein-2 eukaryotic expression vector labeled with green fluorescent protein (GFP).
DESIGN: Single sample observation.
SETTING: Tianjin Hospital.
MATERIALS: The experiment was performed at the Key Laboratory of Hormone and Development, Ministry of Health, Tianjin Medical University from March 2006 to March 2007. pcDNA3.1/CT-hBMP2 plasmid containing full-length hBMP2 gene fragment was provided by Dr. Li; bicistronic eukaryotic expression vector pSELECT-GFPzeo-MCS and Zeo was provided by Invivogen; pTA2?-T Easy by Dingguo, China; restriction enzymes BamHI and NheI, T4 DNA ligase by Jingmei Biotech; PCR upstream and downstream primer synthesis and sequencing by Augct, Beijing.
METHODS: With pcDNA3.1/CT-hBMP2 as template, hBMP2 target fragment was subcloned by PCR binding with designed specific primers. The fragment was bound with pTA2-T-easy and pSELECT-GFPzeo-MCS, separately, and transfected into DH5α cells. pSELECT-GFPzeo-hBMP2 containing GFP was obtained after screening.
MAIN OUTCOME MEASURES: hBMP2 sequence was identified by PCR; whether hBMP2 was cloned into pTA2-hBMP2 and pSELECT-GFPzeo-MCS was identified by digestion and sequencing.
RESULTS: A target fragment of 1 216 bp was obtained by PCR amplification, and cloned into pTA2-T-easy and pSELECT-GFPzeo-MCS. The screening and sequencing results showed that the target fragment was 100% matched with BMP2cDNA sequence (NM-001200) from GenBank.
CONCLUSION: hBMP2 eukaryotic expression vector labeled with green fluorescent protein is successfully constructed.

1Department of Spinal Surgery, Tianjin Hospital, Tianjin 300211, China; 2Department of Pathology, Medi-cal College of Chi-nese People's Armed Police Forces, Tianjin 200162, China; 3Cangzhou Second People's Hospital, Cangzhou 061000, Hebei Province, China

Miao Jun☆, Doctor, Attending physician, Department of Spinal Surgery, Tianjin Hospital, Tianjin 300211, China
mj6688@163.com

Supported by: the National Natural Science Foundation of China, No. 30500516*

Received: 2007-04-20 Accepted: 2007-10-08 (07-50-10-5710/YWY)

Miao J, Liu CR, Huang HC, Xia Q, Shi KS, Cui GS. Constructing bicis-tronic eukaryotic expression plasmid containing human bone morphogenetic protein-2
labeled with green fluorescent pro-tein.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(20):3984-
3987 (China)
[www.zglckf.com/
zglckf/ejournal/
upfiles/08-20/
20k-3984(ps).pdf]

构建含双顺反子绿色荧光蛋白标记人骨诱导形成蛋白2真核表达载体*☆

摘要
背景：腺病毒虽可携带骨诱导形成蛋白2基因转染靶细胞促进其分泌诱导形成蛋白2，但容易出现一些免疫应答反应，以质粒为载体的转染实验应具有良好的前景。
目的：验证构建绿色荧光蛋白标记的人骨诱导形成蛋白2真核表达载体的可行性。
设计：单一样本观察。
单位：天津医院。
材料：实验于2006-03/2007-03在天津医科大学卫生部激素与发育重点实验室（国家级）完成。含完整hBMP2基因片段的pcDNA3.1/CT-人骨诱导形成蛋白2质粒由李曦铭博士惠赠。双顺反子真核表达载体
pSELECT-GFPzeo-MCS为Invivogen公司产品，Zeo购自Invivogen公司。pTA2?-T Easy为鼎国生物技术有限公司产品，限制性内切酶BamHI 和NheI、T4 DNA连接酶（晶美生物工程有限公司），PCR上下游引物合成及测序（北京奥科生物技术有限责任公司）。
方法：以重组质粒pcDNA3.1/CT-人骨诱导形成蛋白2为模板，结合已设计好的特异性引物，采用PCR方法亚克隆出人骨诱导形成蛋白2目的片段，将该片段分别与克隆载体pTA2-T-easy和双顺反子真核表达载体pSELECT-GFPzeo-MCS连接，转化入感受态DH5α细胞中，通过筛选得到含有绿色荧光蛋白的重组表达载体pSELECT-GFPzeo-人骨诱导形成蛋白2。
主要观察指标：采用PCR法鉴定人骨诱导形成蛋白2序列，同时进行酶切及测序鉴定人骨诱导形成蛋白2是否克隆入pTA2-人骨诱导形成蛋白2重组质粒及真核表达载体pSELECT-GFPzeo-MCS中。
结果：PCR获得长度约1 216 bp的目的片段，经与克隆载体pTA2-T-easy和真核表达载体pSELECT-GFPzeo-MCS连接，筛选及序列分析后，证实所插入目的片段与GenBank检索的BMP2cDNA序列（NM-001200）100%匹配。
结论：成功构建含双顺反子绿色荧光蛋白标记人骨诱导形成蛋白2真核表达载体。
关键词：人骨形态发生蛋白2；真核表达载体；绿色荧光蛋白；组织构建

苗 军1，刘春蓉2，黄鸿超1，夏 群1，石可松1，崔国盛3（1天津市天津医院脊柱外科，天津市 300211；2天津武警医学院病理学教研室，天津市 200162；3沧州市第二人民医院，河北省沧州市 061000）
苗 军☆，男， 1974年生，河北省沧州市人，汉族， 2004年解放军总医院毕业，博士，主治医师，主要从事脊柱外科和生物材料的研究。
国家自然科学基金资助(30500516)*

中图分类号: R329.47 文献标识码: A 文章编号: 1673-8225(2008)20-03984-04
苗军，刘春蓉，黄鸿超，群，石可松，崔国盛. 构建含双顺反子绿色荧光蛋白标记人骨诱导形成蛋白2真核表达载体[J].中国组织工程研究与临床康复,2008,12(20):3984-3987
[www.zglckf.com/zglckf/ejournal/upfiles/08- 20/20k-3984(ps).pdf]
(Edited by J. Terrence Jose Jerome/
Su LL/Wang L)