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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,构建真核表达质粒,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突变。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pCIneo中,转染COS-7细胞进行瞬时表达,并用SDS-PAGE和Westernblot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。Westernblot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CD1497～101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。
OBJECTIVE: To alanine substitution mutagenesis of amino acids 97 to 101 in human CD14 signaling transduction promoter region, a eukaryotic expression plasmid was constructed and expressed in COS-7 cells. Methods Two rounds of polymerase chain reaction site-directed mutagenesis was used to mutate 97 to 101 amino acids of N-terminal alanine in human soluble CD14 inferred region of LPS signal transduction. The mutant PCR fragment was cloned into vector pGEM-T for restriction enzyme digestion and sequence analysis. The mutant gene was subcloned into the eukaryotic expression plasmid pCIneo, transfected into COS-7 cells for transient expression, and the expressed protein was detected by SDS-PAGE and Western blot. Results Sequencing results confirmed that there was a continuous alanine substitution mutation in the amino acid 97 to 101 of CD14. SDS-PAGE showed that the mutant gene was transiently expressed in COS-7 cells. Western blot showed that the anti-CD14 polyclonal antibody specifically binds to the recombinantly expressed mutant protein. Conclusion The successful construction and expression of CD1497 ~ 101 amino acid alanine substitution mutants laid the foundation for the study on the biological activity and function of CD14 signaling deletion mutant.