人乳铁蛋白cDNA 基因乳腺表达载体的构建与鉴定

Construction and identification of mammary expressional vector for cDNA of human lactoferrin

摘要:

为了构建人乳铁蛋白基因 (hLF) 的乳腺表达载体并验证其在乳腺细胞中的表达情况，本载体以山羊β-casein基因上游包括启动子、外显子1、内含子1、部分外显子2 作为5′端调控序列，下游包括部分外显子7、内含子7、外显子8、内含子8、外显子9 及3′部分基因组片段作为3′端调控序列，长度分别为6.2 kb 和7.1 kb，将hLF 基因 (目的基因) 和Neo 基因 (筛选标记) 分别插入到5′端调控序列和3′端调控序列的下游，构建成pBC1-hLF

Abstract:

The aim of this study was to construct a mammary gland-specific expressional vector pBC1-hLF-Neo for Human Lactoferrin (hLF) gene and then investigate its expression in the mammary gland epithelium cells. The constructed vector contained the 6.2 kb long 5′ flank regulation region including promoter, other elements and the 7.1 kb long 3′ flank regulation region including transcriptional ending signal of a goat’s β-casein gene. A cassette of Neo gene was also inserted into the vector which gave a total length of 26.736 kb identified by restriction fragment analysis and partial DNA sequencing. The results revealed that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, we transfected the lined vector DNA into the dairy goat′s mammary gland epithelium cells and C127 cells of a mouse′s mammary epithelium by Lipofectamine. After selection with G418 for 8?10 days, G418-risistant clones were obtained. PCR analysis demonstrated that hLF gene cassette had been integrated into the genomic DNA of G418-risistant clones. After proliferation culture, the two kinds of transgenic cells were cultured in serum-free DMEM-F12 medium with prolactin, insulin and hydrocortisone- a medium capable of inducing recombinant hLF expression. RT-PCR, Western blotting and anti-bacteria bioactivity experiments demonstrated that the constructed mammary gland specific vector pBC1-hLF-Neo possessed the desirable bioactivity to efficiently express and could secrete hLF in both mammary gland cells and have the effect of E. coli proliferation inhibition. Paramount to everything, this study laid a firm foundation for preparing the hLF gene transgenic goat fetal-derived fibroblast cells.