人杀菌/通透性增强蛋白活性片段基因在CHO细胞中的表达

Construction of the eukaryotic expression vector of bioactive fragment of human bactericidal/permeability-increasing protein and its expression in CHO cells

目的获得人杀菌/通透性增强蛋白(bactericidal/permeability-increasing protein,BPI)N端活性片段蛋白,为BPI结构和功能的研究提供有力的工具。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BPIN端基因片段,将其克隆入pUC19载体中并进行酶切鉴定和序列测定,然后亚克隆入质粒pcDNA3构建真核表达载体pcDNA-BPI;重组载体通过脂质体转染CHO细胞并筛选阳性克隆,免疫荧光法鉴定重组BPI的表达和免疫学活性。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,与文献报道的序列相比,有6个核苷酸差异。转染筛选的阳性CHO细胞克隆表达产物能够被抗BPI的单克隆抗体所识别。结论BPI活性片段表达载体的构建及真核表达成功,为BPI功能的研究奠定了基础。

AIM： To construct the eukaryotic expression vector of the cDNA sequence encoding bioactive N-terminal fragment of human bactericidaVpermeability-increasing protein （BPI） and express it in CHO cells. METHODS： Total RNA was extracted from human polymorphonuclear leukocytes （PMN） and subjected to reverse transcription, then the human BPI cDNA gene was amplified by nested PCR. The PCR product was cloned into pUC19 plasmid and confirmed by restriction enzyme digestion and DNA sequencing. Then the specific BPI encoding fragment was sub-cloned into pcDNA3 plasmid to form pcDNA-BPIN eukaryotic expression vector. CHO cells were transfected with the recombinant plasmid and the stable clones were selected by G418. The expression of BPI was identified by immunofluorescent assay. RESULTS： Restriction enzyme digestion and DNA sequence analysis revealed that the sequence encoding signal peptide and bioactive N-terminal fragment of BPI had six nucleotide substutions, compared with that of the established human BPI sequence. The expression products of the selected CHO positive cell clones were detected by anti-BPI monoclonal antibody. CONCLUSION： The construction of the eukaryotic expression vector of bioactive fragment of human BPI and its successful expression in CHO cells are helpful to further study of the role of BPI.