CMC-g-DNA接枝共聚物的合成与表征

doi:10.7503/cjcu20140045

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (7): 1590.doi: 10.7503/cjcu20140045

CMC-g-DNA接枝共聚物的合成与表征

李敏, 邢朝晖, 田艳芝, 姜勇()

东南大学化学化工学院, 南京 211189

收稿日期:2014-01-15 出版日期:2014-07-10 发布日期:2014-04-29
作者简介:联系人简介: 姜勇, 男, 博士, 教授, 博士生导师, 主要从事生物大分子研究. E-mail: yj@seu.edu.cn
基金资助:
国家自然科学基金(批准号: 21174029)和教育部留学回国人员科研启动基金资助

Preparation and Characterization of the CMC-g-DNAHybrid Copolymers^†

LI Min, XING Zhaohui, TIAN Yanzhi, JIANG Yong^*()

School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China

Received:2014-01-15 Online:2014-07-10 Published:2014-04-29
Contact: JIANG Yong E-mail:yj@seu.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China(No21174029) and the Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, China

摘要/Abstract

摘要：

在磷酸盐缓冲溶液中用1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)与N-羟基琥珀酰亚胺(NHS)活化羧甲基纤维素钠(CMC)侧链上的羧基; 在室温下再将活化的CMC与5'端经氨基修饰的单链脱氧核糖核酸(DNA)齐聚物(ODNs)反应, 获得CMC上接枝ODNs的共聚物(CMC-g-ODNs), 以Lambda DNA为模板, 通过聚合酶链式反应(PCR), 将接枝的ODNs扩增为长度为1300个碱基对的双链DNA, 从而制得CMC侧链上接枝DNA的共聚物CMC-g-DNA. 采用傅里叶红外光谱仪测定CMC与NHS形成的中间体; 用水平式琼脂糖凝胶电泳和垂直板变性聚丙烯酰胺凝胶电泳对CMC-g-DNA接枝共聚物进行表征. 结果表明, 合成了CMC-g-DNA接枝共聚物, 且在酸性条件下CMC的活化效果更好; 同时, 接枝在CMC上的DNA在琼脂糖凝胶电泳中迁移速率加快, 而在聚丙烯酰胺凝胶电泳中迁移速率减慢.

关键词: 脱氧核糖核酸(DNA), 羧甲基纤维素钠, 接枝共聚物, 凝胶电泳, 聚合酶链式反应, 迁移速率, 基因载体

Abstract:

Sodium carboxymethyl cellulose grafted DNA(CMC-g-DNA) hybrid copolymer, which can be a potential gene drug, was synthesized and characterized by both chemical and biological methods. First, the carboxyl groups on the side chain of CMC were activated by both 1-(3-dimethylaminopropyl)-3-ethylcarbo-diimide(EDC) and N-hydroxy-succinimide(NHS) in phosphate buffer at both neutral and acid condition, respectively. After that, the CMC-NHS reacted with amino-functionalized oligodeoxynucleotides(NH₂-ODNs) to get CMC-g-ODNs grafted copolymer. Then CMC-g-ODNs were used as forward primers for the next-step polymerase chain reaction(PCR) to generate the final CMC-g-DNA hybrid copolymer. The infrared spectrum detection proved that the side carboxyl groups of the CMC were substituted very well by NHS and the efficiency was higher at acid condition than neutral. The obtained CMC-g-DNA copolymers were characterized by agarose gel electrophoresis and polyacrylamide gel electrophoresis, respectively. And the results showed that DNA was grafted on CMC successfully. An interesting finding is that CMC-g-DNA moved faster than control DNA in agarose gel electrophoresis, while it migrated slower than control polyacrylamide gel electrophoresis.

Key words: DNA, Sodium carboxymethyl cellulose(CMC), Hybrid copolymer, Gel electrophoresis, Polymerase chain reaction(PCR), Migrated speed, Gene combination

中图分类号:

O636.9

TrendMD:

李敏, 邢朝晖, 田艳芝, 姜勇. CMC-g-DNA接枝共聚物的合成与表征. 高等学校化学学报, 2014, 35(7): 1590.

LI Min, XING Zhaohui, TIAN Yanzhi, JIANG Yong. Preparation and Characterization of the CMC-g-DNAHybrid Copolymers^†. Chem. J. Chinese Universities, 2014, 35(7): 1590.

图/表 5

Scheme 1 S ynthetic routes of CMC-g-DNA

Fig.1 Polymerase Chain Reaction(PCR) as a tool for the preparation of CMC-g-DNA

Fig.2 FTIR spectra of CMC(a), activated CMC at pH=7.2(b) and activated CMC at pH=6.5(c)

Fig.3 Picture of 3% horizontal agarose gel electrophoresis of 1000 bp DNA Marker P(lane 1), unreacted ODNs(lane 2), CMC-g-ODNs(lane 3, CMC activated at pH=7.2) and CMC-g-ODNs(lane 4, CMC activated at pH=6.5)

Fig.4 Picture of 1% horizontal agarose gel electrophoresis(A) and 5% vertical polyacrylamide gel electrophoresis(B) (A) Lane 1: 1000 bp DNA Ladder; lane 2: 1300 bp DNA; lane 3: CMC-g-DNA; lane 4: CMC-g-ODNs. (B) Lane 1: 1300 bp DNA; lane 2: CMC-g-DNA.

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CMC-g-DNA接枝共聚物的合成与表征

Preparation and Characterization of the CMC-g-DNAHybrid Copolymers^†

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CMC-g-DNA接枝共聚物的合成与表征

Preparation and Characterization of the CMC-g-DNAHybrid Copolymers†

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Preparation and Characterization of the CMC-g-DNAHybrid Copolymers^†