人α-防御素-1(α-HNP-1)基因真核表达载体的构建及在大鼠骨髓间充质干细胞中的转染表达

Construction the Eukaryotic Expression Vector of Human α-Defensin-1(α-HNP-1) Gene and Transfect to Rat Marrow Mesenchymal Stem Cells

目的构建人α-防御素-1(α-HNP-1)基因的真核表达载体,并且转染大鼠骨髓间充质干细胞。方法提取人外周血粒细胞中的总RNA,反转录为cDNA作为模板,采用PCR的方法扩增得到人α-防御素-1(α-HNP-1)的基因片断。将扩增产物连接入pGEM-T载体,转化大肠杆菌DH5α感受态细胞,蓝白筛选,对PCR及酶切鉴定含有目的片断的克隆进行测序。经测序证实无误后,将获得的pGEM-T-HNP-1重组质粒上的α-HNP-1基因亚克隆到真核表达载体pcDNA3.1(-)上,构建HNP-1的真核表达载体pcDNA3.1-HNP-1。将真核表达载体pcDNA3.1-HNP-1用脂质体法转染原代大鼠骨髓间充质干细胞,用免疫组化法检测α-HNP-1的表达。结果获得预期大小为303bp的RT-PCR产物;经PCR、酶切鉴定和DNA测序分析证实重组质粒载体pcDNA3.1-HNP-1构建正确;免疫组化法显示转染细胞呈阳性反应。结论成功构建HNP-1基因的真核表达载体,并且在大鼠骨髓间充质干细胞中能成功表达。

Objective: To construct an eukaryotic expression vector containing the gene encoding human α-defensin-1(α-HNP-1) and transfect it to rat marrow mesenchymal stem cells.Method: Total RNA of human neutrophil in peripheral blood was extracted and reverse transcribed into complementary DNA which was used as template for the polymerase chain reaction for obtaining α-HNP-1 gene. PCR product of expected size was inserted into pGEM-T vector and then transformed into Escherichia coli DH5α competent cell. The clone containing the DNA fragment was identified by PCR and restriction analysis. After analyzed by sequencing, the recombinant pGEM-T-HNP-1 plasmid was digested with EcoRⅠ and BamHⅠ,and subcloned into eukaryotic plasmid pcDNA3.1(-) to construct recombinant plasmid pcDNA3.1-HNP-1. The plasmid vector pcDNA3.1-HNP-1 then transfected to rat marrow mesenchymal stem cells by lipofection reagent. Then used the immunocytochemistry to detect expression of α-HNP-1 gene.Results: We obtained a 303bp DNA fragment which is identical to the α-HNP-1 cDNA sequence reported in the GenBank; PCR,restriction enzyme digesting and DNA sequencing confirmed the recombinant eukaryotic plasmid vector which contained α-HNP-1 gene had been constructed correctly. And the transfected cells showed positive staining for α-HNP-1 by immunohistochemistry assay.Conclusion: An eukaryotic expression vector pcDNA3.1-HNP-1 was constructed successfully, and α-HNP-1 gene could be transfected into rat marrow mesenchymal stem cells and expressed by using liposome.