人CD226分子胞膜外区D1和D2真核表达载体的构建、表达和鉴定

Construction and expression of human CD226 extracellular domain 1 and domain 2 gene eukaryotic expression vectors and identification of the fusion proteins

目的构建含有人CD226分子胞膜外区结构域1(D1)和结构域2(D2)的真核表达载体,表达并纯化重组蛋白。方法用特异性引物分别扩增CD226D1和D2的基因,定向插入真核表达载体pSecTag2B-Fc,进行酶切和DNA测序鉴定,重组质粒瞬时转染293T细胞,收集上清,纯化蛋白及SDS-PAGE和Western blot鉴定表达产物。结果利用PCR方法克隆出CD226胞膜外区D1和D2的基因,并正确插入pSecTag2B-Fc载体中,真核表达载体瞬时转染293T细胞,Western blot结果证实转染293T细胞的培养上清液中含有hCD226D1-Fc和hCD226D2-Fc融合蛋白,培养上清经亲和层析柱纯化,可获得较高纯度的重组蛋白。结论成功的构建了分泌型融合蛋白hCD226D1-Fc和hCD226D2-Fc真核表达载体,获得纯度较高的融合蛋白,为进一步探讨其配受体的相互作用机制奠定了基础。

Objective To construct and express eukaryotie expression vectors of domain 1 and domain 2 in the extraeellular region of human CD226 and identify their expressed fusion proteins.Methods The hCD226D1 and hCD226D2 genes were amplified from the full length CD226-flag and cloned to pMD18-T vector by PCR. The recombinant plasmids were digested and inserted into the same site of expression vector pSecTag2B to construct pSecTag2B/hCD226D1 and pSecTag2B/hCD226D2. The recombinant plasmids were digested and subjected to gene sequencing before recombinant expression vectors were transfected into 293T cells. The expressed fusion proteins hCD226D1-Fc and hCD226D2- Fc were purified by WT6 conjugated Sepherese-4B affinity column, and analyzed by SDS-PAGE and Western blotting. Results The D1 and D2 genes of hCD226 were successfully cloned by PCR. And the sequence of hCD226D1-Fc and hCD226D2-Fc genes＇ ORF were consistent with the sequence of GenBank. Digestion analysis showed a band of about 350 bp. CD226 D1-Fc and D2-Fc fusion proteins were confirmed by SDSPAGE and Western blotting. Csmehrsion hCD226D1-Fc and hCD226D2-Fc fusion proteins are expressed successfully, which will provide the foundation for further study on the interaction of CD226 with its ligands.