【摘要】 目的:构建牛催乳素(bPRL)为目的基因转基因动物-乳腺生物反应器。方法:采用长距离PCR技术,首次从正常牛外周血细胞中克隆到全长9 388 bp的bPRL基因组序列,利用亚克隆的方法将bPRL基因组DNA克隆到真核表达载体pcD-NA3.1(+)上,构建出pcDNA3.1(+)/bPRL的重组表达载体,通过脂质体转染至非洲绿毛猴肾细胞株(COS-7),逆转录-PCR得到长度为804 bp扩增片段。结果:序列测定bPRL基因包括全部5个外显子和4个内含子,5’端854 bp的上游调控区以及3’端69 bp的非翻译区(UTR)。bPRL cDNA包含信号肽在内的全长序列。结论:克隆的bPRL基因组DNA序列在转录水平上具有生物学功能,在体外培养的真核细胞中能够进行正常表达,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。更多还原

【Abstract】 Objective: To construct the bPRL transgentic animals and its mammary gland bioreactor. Methods: A full-length sequence of bovine prolactin (bPRL) genomic DNA with 9 388 bp was amplified by long polymerase chain reaction (Long PCR) from white blood cells of normal bovine. The bPRL genomic DNA was inserted into mammalian expression plasmid pcDNA3. 1 ( + ) and constructed pcDNA ( + ) /bPRL recombinant eukaryotic expression vector. The recombinant plasmid was transfected into COS - 7 cells with lipofectin,and 804 bp bPRL cDNA was amplified by RT-PCR. Results: Through DNA sequence analysis, the bPRL genomic fragment was proved to consisted of 5 exons,4 introns,854 bp of 5’ upstream regulatory region and 69 bp of 3’ UTR; and it contained a full-length sequence with the signal peptide. Conclusion: It was indicated that the bPRL genomic DNA had its biological function at the transcripti nal level, it could normally express in eukaryocytes of culture in vitro, and lay a solid foundation for constructing transgentic animals and its mammary gland bioreactor.更多还原