摘要
目的:检测β酪蛋白基因5′侧序列的启动BglⅡ效果及构建真核表达载体pcDNA3.165.方法:采用SalⅠ和BamHⅠ对pMDT/65双酶切,得到6465bp的β酪蛋白基因5′侧序列;将启动子片段插入到XhoⅠ和BglⅡ双酶切后的pGL3Basic载体里;获得重组报告基因载体pGL3B65.瞬时转染西农萨能奶山羊原代培养的乳腺上皮细胞,检测β酪蛋白基因5′侧序列的启动效果.将β酪蛋白基因5′侧序列插入真核表达载体pcDNA3.1(-)的XhoⅠ和BamHⅠ位点之间,从而获得载体pcDNA3.165.结果:长度为6465bp的β酪蛋白基因5′侧序列能有效地启动荧光素酶报告基因,并正确构建了真核表达载体pcDNA3.165.结论:克隆的β酪蛋白基因5′侧序列在转录水平上具有生物学功能,为进一步建立转基因动物乳腺生物反应器奠定了坚实的基础.
AIM: To detect the promote effect of β-casein gene 5′ flanking sequence and to construct universal expression vector pcDNA3.1-65. METHODS: ① After the β-casein gene 5′ flanking sequence with 6465 bp from pMD-T/65 vector was double digested with [WTFX]SalⅠ/ [WTFX]BamHⅠ, the 6465 bp sequence was obtained.And 6465 bp sequence was inserted into pGL3-Basic vector which had been double digested with XhoⅠ/BglⅡ.The new clone was named pGL3-B65.② Detect the activity of pGL3-B65 constructed by transient transfection in primary goat mammary gland epithelial cells.③ Insert the β-casein gene 5′ flanking sequence into [WTFX]XhoⅠ /[WTFX]BamHⅠ of the pcDNA3.1 vector to construct pcDNA3.1-65 vector. RESULTS: The β-casein gene 5′ flanking sequence with 6465 bp sequence could promote luciferase reporter gene effectively. The pcDNA3.1-65 vector was well constructed. CONCLUSION: It indicates that the cloned β-casein gene 5′ flanking sequence would have its biological function at the transcriptional level, and lay a solid foundation for constructing transgenic animals and its mammary gland bioreactor.
出处
《第四军医大学学报》
北大核心
2005年第12期1066-1069,共4页
Journal of the Fourth Military Medical University