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Construction of eucaryotic expression vector of bioactive fragment gene of human bactericidal/permeability-increasing protein and its expression in COS-7 cells
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 4
- DOI:
- 10.3321/j.issn:1000-2790.2000.04.041
- Key Word:
- 杀菌/通透性增加蛋白;逆转录PCR;序列分析;基因重组;真核表达
Abstract： 目的构建人杀菌/通透性增加蛋白(BPI)N端活性片段cDNA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 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.
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