乔录新1 张世杰2 石英1 陈德喜1△.基于同尾酶技术构建CCL3L1 基因串联重组质粒的方法[J].,2012,12(2):239-241 |
基于同尾酶技术构建CCL3L1 基因串联重组质粒的方法 |
A Novel Method for Constructing Recombinant Plasmid of CCL3L1Tandem Repeats Via Isocaudamer Technology |
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DOI: |
中文关键词: 同尾酶 CCL3L1 基因串联 |
英文关键词: Isocaudamer CCL3L1 Tandem repeats |
基金项目:国家自然科学基金面上项目(30870853);国家自然科学基金国际(地区)合作交流项目(30910103915); |
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中文摘要: |
目的:利用同尾酶技术将CCL3L1 基因重复连续插入pcDNA6.2-GW/miR 载体,构建含有CCL3L1 基因串联体的重组质粒,
实现小片段CCL3L1 有效延长。方法:PCR 扩增CCL3L1 基因并在引物的两端设有同尾酶BamHI 和BglII 限制性内切酶位点,纯
化PCR 产物插入pMD18-T 载体,阳性克隆命名为pMD18T-CCL3L1。BamHI 和BglII 双酶切pMD18T-CCL3L1 和pcDNA6.
2-GW/miR 载体后将第一个CCL3L1 片段插入pcDNA6.2-GW/miR 载体命名为pcDNA6.2-CCL3L1-1。由于载体本身在BglII 位点
后带有XhoI 酶切位点利用BamHI 和XhoI 切割pcDNA6.2-CCL3L1-1 回收CCL3L1 片段,BglII 和XhoI 切割pcDNA6.
2-CCL3L1-1 回收大片段做载体重组形成含有两个连续CCL3L1 片段的质粒命名为pcDNA6.2-CCL3L1-2,重复此步骤可得到含
有N 个CCL3L1 基因串联体的重组质粒pcDNA6.2-CCL3L1-X。结果:经酶切和测序证实成功构建含有4 个CCL3L1 基因串联体
的重组质粒pcDNA6.2-CCL3L1-4,并同时产生含有1 个和2 个CCL3L1 基因串联体的重组质粒。结论:利用同尾酶技术可以快速
有效地构建CCL3L1 基因串联重组质粒,实现目的片段的无限扩大,为小片段基因表达的研究奠定基础。 |
英文摘要: |
Objective: To construct the recombinant plasmid containing CCL3L1 tandem repeats by continuous and repeated inserting
CCL3L1 gene into pcDNA6.2-GW/miR vector by isocaudamer technology, resulting in effective extension of a small fragment of
CCL3L1. Methods: Firstly, CCL3L1 gene was amplified by using the primers with two ends of BamHI and BglII restriction enzyme sites,
then the purified PCR products were inserted into pMD18-T vector. Secondly, the positive clone products, named pMD18T-CCL3L1,
and pcDNA6.2-GW/miR were digested by BamHI and BglII. Then the first CCL3L1 fragment was inserted into pcDNA6.2-GW/miR,
and the correct plasmid was named pcDNA6.2-CCL3L1-1. There was a XhoI behind the BglII in the vector, so pcDNA6.2-CCL3L1-1
could be digested by XhoI and BglII to obtian linear CCL3L1, then pcDNA6.2-CCL3L1-1 was digested by BamHI and BglII to obtain
linear vector. The two fragments were ligated and the correct plasmid which contained two consecutive CCL3L1 fragments was named
pcDNA6.2-CCL3L1-2. Repeating this step, the recombinant plasmid pcDNA6.2-CCL3L1 -X which contained N-series body of CCL3L1
gene could be obtained. Results: Enzyme digestion and sequencing confirmed that the recombinant plasmid pcDNA6.2-CCL3L1-4 was
successfully constructed, which contained four consecutive CCL3L1 fragments, and also produced and two recombinant plasmids containing
1or 2 CCL3L1 tandem repeats. Conclusion: The recombinant plasmid of CCL3L1 tandem repeats would be constructed quickly
and efficiently with Isocaudamer. A method for unlimited expansion of the fragment was established, providing a basis for the study
about small fragments' expression. |
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