可溶性CD14突变体的构建与表达研究

目的 将人CD14信号转导启动区97－101氨基酸进行丙氨酸置换突变。，构建直核表达质粒，并在COS－7细胞中进行表达。方法 采用两轮聚合酶链反应定点突变方法，对人可溶性CD14推测中的LPS信号转导启动区之一，N端97－101氨基酸用丙氨酸进行置换交换。将突变PCR片段克隆至载体pGEM-T,进行酶切鉴定及序列分析。将突变基因亚克隆至真核表达质粒pClneo中，转染COS－7细胞进行瞬时表达，并用SDS－PAGE和Western blot检测表达蛋白。结果 测序结果证实，CD14的N端97－101氨基酸发生了连续丙氨酸置换突变。SDS－PAGE表明，突变体基因在COS－7细胞中得到瞬时表达。Western blot显示，抗CD14多克隆抗体可与重组表达的突变体蛋白特异性结合。结论 成功地构建并表达CD14 97－101氨基酸丙氨酸置换突变体，为研究CD14信号转导缺失突变体的生物学活性与功能打下了基础。

Construction and expression of soluble CD14 of mutant

Aim To cause the putative signaling domain 97 101aa of N terminal 1 152aa of human CD14 to produce alanine substitution mutation and to construct eukaryotic expression plasmid, and then express in COS 7cells. Methods Alanine substitution was introducd into the N terminal 97 101 amino acids of human soluble CD14 truncated at amino acid of N terminal 152aa, by double rounds of PCR. 97 101aa region was one of the putative LPS signaling domain of the protein CD14. Mutant fragment obtained was cloned into vector pGEM T, and identified by endonucleases digestion and sequencing. Then the mutant CD14 gene was subcloned into plasmid pCIneo. The constructed recombinant expression plasmid was transfected into COS 7 cells. Results The sequencing showed alanines were introduced into 97 101aa of N terminal 1 152aa of CD14. SDS PAGE showed the mutant gene has been expressed in COS 7cells. Relative molecular mass(Mr) of expressed protein was about 26 000. Western blot analysis indicated the mutant protein could be recognized by anti CD14 polyclonal antibody. Conclusion The CD14(97 101)A152aa gene is constructed. Mutant protein has been expressed successfully in COS 7 cells. The above results lay the foundation for further studying biological activity of signaling deletion mutant CD14.