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目的 构建人杀菌 /通透性增加蛋白 (BPI) N端活性片段 c DNA序列的真核表达载体并了解其在 COS- 7细胞中的表达 .方法 提取人外周血多形核白细胞 (PMN)总 RNA进行逆转录反应 ,经套式 PCR扩增出 BPI N端基因片段 ,将其克隆入 p UC19载体中并进行酶切鉴定和序列测定 ,然后亚克隆入质粒 pc DNA3构建真核表达载体 pc DNA- BPI;重组载体通过脂质体转染 COS- 7细胞 ,免疫荧光法鉴定 BPI的表达 .结果 酶切鉴定和序列分析证实重组质粒含有包括信号肽在内的 BPI活性片段编码序列 ,转染实验表明重组质粒能在COS- 7中表达出具有活性的 BPI片段 .结论 BPI活性片段真核表达载体构建及表达成功 ,为下一步 BPI功能的研究奠定了基础
Objective To construct a eukaryotic expression vector for human bactericidal/permeability-increasing protein (BPI) N-terminal active fragment c DNA sequence and to understand its expression in COS-7 cells. Methods Total human peripheral blood polymorphonuclear leukocyte (PMN) was extracted. The reverse transcription reaction was performed on RNA and the BPI N-terminal gene fragment was amplified by nested PCR, cloned into the pUC19 vector and subjected to restriction enzyme digestion and sequence analysis. The recombinant plasmid was then subcloned into the plasmid pc DNA3 to construct the eukaryotic expression vector pc DNA. - BPI; Recombinant vector was transfected into COS-7 cells by lipofectamine and the expression of BPI was identified by immunofluorescence. Results The restriction enzyme digestion and sequence analysis confirmed that the recombinant plasmid contains the coding sequence of the BPI active fragment including the signal peptide, transfection experiments. The result showed that the recombinant plasmid could express active BPI fragment in COS-7. Conclusion The construction and expression of the eukaryotic expression vector of BPI active fragment laid a foundation for the further study of BPI function.