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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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