【摘要】 目的 :将人可溶性 CD14 重组基因 ,用真核表达系统进行表达 ,为进一步研究 CD14 的功能打下基础。方法 :用 PCR方法扩增 s CD14 34 8aa片段 ,将产物与载体 p EF1/ His C酶切 ,纯化 ,连接后克隆至大肠杆菌 DH5α中 ,酶切鉴定 ;将重组质粒 p EF1/ His C/ s CD14 34 8aa用脂质体转染法转染至 CHO细胞中 ,用SDS- PAGE、Westen- Blot方法进行检测。结果 :阳性重组子可以切出 10 4 4 bp大小的片段 ,得到重组质粒 p EF1/ His C/ s CD14 34 8aa;转染后的 CHO细胞裂解液 ,Westen- Blot在大小约 530 0 0附近有一条较强的特异性棕色条带 ,说明 s CD14 34 8aa基因在 CHO细胞中得到表达。在 CD14 的 N端有 5个潜在的糖基化位点 ,经真核系统表达 ,表达产物分子量与文献报道基本一致 ,证明重组蛋白糖基化完全。结论 :重组质粒p EF1/ His C/ s CD14 在真核细胞中得到表达更多还原

【Abstract】 Objective:In order to lay foundation on further studying the function of CD 14 , the human soluble CD 14 recombinant gene was expressed by eukayotic expressing system. Methods:After sCD 14 348aa segment which was amplified by PCR and pEF1/HisC vactor were digested, purified and ligated, the recombinant pEF1/His/sCD 14 348aa was cloned to E coli DH5α and was authenticated by digestion. The recombinant pEF1/His/sCD 14 348aa was transfected to CHO by lipseme, which was assayed with SDS PAGE and Western blotting. Results:The recombinant pEF1/His/sCD 14 348aa of 1 044 bp was obtained from poeitine recombinant by digestion. The transfected CHO cell lysis liquild was performed by western blotting analysis and a more intensive specific brown band could be observed on appoximately 53 000,suggesting that sCD 14 348aa was expressed in CHO cells. The size of expressed product of sCD 14 348aa with eukaryotic systems was consistent with that reported on literatures. Conclusion: The recombinant plasmid pEF1/His/sCD 14 was expressed in eukaryotic cell. [更多还原