人源抗菌肽LL-37/hCAP-18真核表达载体的构建及其在人乳腺癌细胞MCF-7中的表达

Construction of Eukaryotic Expression Vector for LL-37/hCAP-18 and Its Expression in Human Breast Cancer MCF-7 Cells

目的构建人源抗菌肽LL-37/hCAP-18的真核表达载体,并在人乳腺癌细胞MCF-7中进行表达。方法以重组质粒pcDNA4.0-LL-37为模板,采用PCR法扩增LL-37/hCAP-18基因,连入pEGFP-c1质粒,构建真核表达载体pEGFP-c1-LL-37,瞬时转染人乳腺癌细胞MCF-7,采用激光共聚焦显微镜观察重组质粒的转染效率,RT-PCR法检测目的基因的表达。结果所构建的抗菌肽LL-37/hCAP-18真核表达载体经双酶切和测序证明构建正确,在瞬时转染MCF-7细胞48h后,目的基因转染效率较高,约为40%,RT-PCR结果显示,重组质粒转染的细胞可扩增出339bp的目的基因条带。结论已成功构建了人源抗菌肽LL-37/hCAP-18的真核表达载体,并在人乳腺癌细胞中获得表达,为进一步研究其抗肿瘤作用奠定了基础。

Objective To construct a eukaryotic expression vector for LL-37/hCAP-18 （18 kD human cationic antimicrobial peptide） and express in human breast cancer MCF-7 cells. Methods Amplify LL-37/hCAP-18 gene by PCR using recombinant plasmid pcDNA4. 0-LL-37 as a template and insert into plasmid pEGFP-c1. Transfect MCF-7 cells transiently with the constructed recombinant plasmid pEGFP-cl-LL-37, observe the transfection efficacy by laser co-focal microscopy, and determine the expression of target gene by RT-PCR. Results Both restriction analysis and sequencing proved that eukaryotic expression vector pEGFP-cl-LL-37 was constructed correctly. The transfection efficacy of target gene in MCF-7 cells 48 h after transient transfection was about 40%. The target gene band at a length of 339 bp was amplified from transfeeted MCF-7 cells. Conclusion The eukaryotic expression vector for LL-37/hCAP-18 was successfully constructed and expressed in human breast cancer MCF-7 ceils, which laid a foundation of further study on the antitumor effect of LL-37/hCAP-18.