人CD134L基因真核细胞表达载体的构建、鉴定及序列分析

Construction and identification of the recombinant mammalian cell expression plasmid for the expression of the human CD134L cDNA

目的 构建人CD1 34L真核细胞表达载体并对其进行序列分析。方法 采用PCR方法扩增人CD1 34L基因cDNA ,先后经BamHI和XhoI酶切并纯化 ,再定向克隆到高效真核表达载体pcDNA3中 ,构建成pcDNA3 CD1 34L ,通过PCR扩增、双酶切后琼脂糖电泳分析及序列测定进行鉴定。结果 pcDNA3 CD1 34L经BamHI和XhoI双酶切后出现两条目的条带、经PCR扩增出现单一目的条带、序列测定结果与Genebank的序列完全一致。结论 成功地克隆了人CD1 34L基因cDNA ,构建了真核表达载体 ,本研究为探讨CD1 34L与淋巴细胞活化的关系。

Objective To construct a recombinant mammalian cell expression plasmid for the expression of human CD134L cDNA. Methods A 596bp cDNA fragment was amplified by PCR method from plasmid of PGEX 4T1 CD134L. The fragment was cloned into the pcDNA3 vector. The cloned insert was verified by double digestion with BamHI and XhoI and sequenced.Results A 596bp DNA fragment was amplified and the DNA sequence was identical to the published sequence encoding hCD134L gene.Conclusion The recombinant mammalian cell expression plasmid, pcDNA3 hCD134L, was successfully constructed. This recombinant plasmid can be used for the further studies of the functions of the hCD134L gene.