基于同尾酶技术构建CCL3L1 基因串联重组质粒的方法

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。

A Novel Method for Constructing Recombinant Plasmid of CCL3L1 Tandem Repeats Via Isocaudamer Technology

Objective: To construct the recombinant plasmid containing CCL3L1 tandem repeats by continuous and repeated in-serting 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 con-taining 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.