人中性粒细胞多肽1,3真核表达载体的构建与鉴定

目的：扩增人中性粒细胞多肽1，3(HNP1，3)基因片段，构建真核表达载体pcDNA3．1／V5-His-TOPO／HNP1，3。方法：从健康人中性粒细胞中提取总RNA，用RT-PCR方法扩增出HNP1，3基因完整的cDNA片段，应用TA克隆插入pMD18-T载体，经酶切鉴定及序列分析确证后，将其亚克隆至真核表达载体pcDNA3．1／V5-His-TOPO中。结果：从人中性粒细胞中克隆出人HNPl1，3基因，经测序分析与GenBank公布的人HNP1，3核苷酸序列同源性为100％，成功地构建了真核表达载体pcDNA3．1／V5-His-TOPO／HNP1，3。结论：真核表达载体pcDNA3．1／V5-His-TOPO／HNP1，3的成功构建，为后续重组基因的真核／表达及其对HIV-1抑制作用的研究奠定了实验基础。

Construction and identification of human neutrophil peptide eukaryotic expression vector

Objective:To obtain encoding gene segment of human neutrophil peptides (HNP1,3) and construct the eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO- HNP1,3. Methods:RNA was extracted from human neutrophil cell as template, cDNA encoding mature HNP1,3 was amplified by RT-PCR and inserted into cloning vector pMD18-T. After restriction endonuclease digestion and DNA sequencing confirmation the gene was subcloned into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO. Results:The gene encoding HNP1,3 was cloned whose DNA sequence was 100% identical with that published in GenBank, a recombinant expression plasmid pcDNA3.1/V5-His-TOPO/HNP1,3 was successfully constructed. Conclusion:The successful construction of pcDNA3.1/V5-His- TOPO/HNP1,3 lies a basis for eukaryotic expression and further research on anti-HIV activity of HNP1,3.