人源阳离子抗菌肽hCAP-18真核表达载体的构建与表达

Construction of Eukaryotic Expression Vector for Human Cationic Antimicrobial Peptide(hCAP-18)

目的构建人源性阳离子抗菌肽hCAP-18的真核表达载体pcDNA4/Myc-His-hCAP-18,转染真核细胞,并检测其表达。方法以正常人外周血中性粒细胞的总RNA为模板,用RT-PCR技术钓取hCAP-18编码序列的全长cDNA,并克隆至pcDNA4/Myc-His真核表达载体上,转染真核细胞株HeLa。RT-PCR及Western blot方法检测目的基因的表达。结果所构建的真核表达载体pcDNA4/Myc-His-hCAP-18,经酶切鉴定所释放片段大小与预期相符,基因序列与GenBank上登录的序列一致,目的基因在转染细胞中获得高水平表达。结论已成功构建真核表达载体pcDNA4/Myc-His-hCAP-18,并在转染细胞中高效表达。

Objective To construct a eukaryotic expression vector for human cationic antimicrobial pepfide（hCAP-18） and determine the expressed product. Methods Amplify the full-length eDNA encoding hCAP-18 by RT-PCR using the total RNA of neutrophils of healthy human peripheral blood as template and clone into eukaryotie expression vector pcDNA4/Mye-His. Transfect the construeted recombinant plasmid pcDNA4/Mye-His-hCAP-18 to HeLa cells ,identify the target gene by RT-PCR and the expressed product by Western blot. Results The length of cloned gene fragment was consistent with that expected, and its sequence was identical to that reported in GenBank. The recombinant protein with a relative molecular weight of 20 000 was expressed in transfected cells. Conclusion Eukaryotie expression vector pcDNA4/Mye-His-hCAP-18 was successfully constructed,and hCAP-18 was highly expressed in transfected cells.