【作者】 张曼；

【导师】 郑月茂；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2015， 硕士

【摘要】 乳腺生物反应器是用乳蛋白基因启动子和调控区指导外源基因在动物乳腺中定位表达。乳腺生物反应器的应用价值与外源基因表达水平的高低相关。乳腺生物反应器制备的关键是乳腺特异性表达载体的构建,而启动子的选择是乳腺表达载体构建的核心。为了使外源基因在转基因动物乳腺中能够高效特异地表达,本实验以西农萨能奶山羊β-乳球蛋白基因为研究对象,扩增了山羊的5ˊ侧翼区,内含子1、内含子2和3ˊ端调控区域序列,构建了真核表达载体,初步探讨了该启动子的活性和表达特异性。结果如下:(1)利用高保真PCR方法克隆了奶山羊β-乳球蛋白基因启动子(2.1 kb,包括5’侧翼区和部分第一外显子),3’端调控成分(最后一个不翻译外显子,内含子和3’侧翼区)及第一内含子和第二内含子。测序分析表明,与其他反刍动物相比较,奶山羊BLG基因5’端调控序列保守性优于3’端调控成分。(2)为了筛选出启动效率高的启动子片段,在下游引物相同的条件下,以克隆的β-乳球蛋白基因2.1 kb启动子为模板,进行删减分析。分别扩增了2081 bp(﹣2041/+40),1981 bp(﹣1941/+40),1881 bp(﹣1841/+40),1681 bp(﹣1641/+40),1481 bp(﹣1441/+40),1281 bp(﹣1241/+40),1081 bp(﹣1041/+40),881 bp(﹣841/+40),681 bp(﹣641/+40),481 bp(﹣441/+40),431 bp(﹣391/+40),381 bp(﹣341/+40)和331 bp(﹣291/+40)共13个片段,将其分别插入质粒PGL4.10的多克隆位点,并分别转染HC11、293T和山羊胎儿成纤维细胞,通过双荧光素酶报告基因检测发现,山羊BLG基因的启动子具有组织特异性,片段大小为431 bp(﹣391/+40)时启动子活性最高。(3)为了探究内含子在基因表达中的作用,我们以pEGFP-C1为骨架载体,通过AseⅠ和NheⅠ双酶切,用431 bp(﹣391/+40)启动子替换pEGFP-C1的启动子CMV,以克隆的内含子1、内含子2和3ˊUTR为调控元件构建载体pE-BLGU11,pE-BLGIN1U11和pE-BLGINU。通过qRT-PCR和Western blot,在mRNA和蛋白质表达水平上检测内含子在基因表达中的作用。结果显示BLG基因的内含子具有增强基因表达的作用,且第一内含子的作用比第二内含子作用更为明显。本实验通过PCR克隆并分析了奶山羊β-乳球蛋白基因的启动子序列,通过梯度删减筛选出启动效率最高的启动子区域;以pEGFP-C1为骨架,证明了β-乳球蛋白基因的内含子具有增强子的作用。利用克隆的调控元件构建了山羊乳腺特异性表达载体,为提高外源基因在乳腺组织中的表达及乳腺生物反应器的制备奠定了基础。更多还原

【Abstract】 Mammary gland bioreactor is made of milk protein gene promoter and regulatory region to guide exogenous gene expression in mammary gland. The quality of mammary gland bioreactor directly related to the level of exogenous genes. The key to mammary gland bioreactor is specific construction of expression vector, and promoter play an important role in construction of mammary gland expression vector. In order to realize the high and specific expression of foreign genes in transgenic animal mammary gland, this study using gene recombination technology, amplified the 5 ˊ-flank, intron 1, intron 2 and 3 ˊ UTR fromβ-lactoglobulin gene of saanen dairy goats, and constructed the eukaryotic expression vector,as the preliminary exploration of the promoter activity and expression of specificity.(1) We cloned the promoter of β-lactoglobulin gene by high fidelity polymerase chain reaction(PCR)(2.1 KB, including 5’ flanking region and part of the first exon), 3’UTR(the last exon, intron 6 and 3’flanking region) and the first and second introns. Sequencing analysis showed that the sequence conservative of 5’ flanking region of dairy goat BLG gene is superior to the 3’ end control components compared to other ruminants.(2) To sift the high efficiency of the promoter fragment, we amplified 13 segments from the promoter of BLG 2.1 kb, which are 2081 bp(-2041/+40), 1981 bp(-1941/+40), 1881 bp(-1841/+40), 1681 bp(-1641/+40), 1481 bp(-1441/+40), 1281 bp(-1241/+40), 1081 bp(-1041/+40), 881 bp(-841/+40), 681 bp(-641/+40), 481 bp(-441/+40), 431 bp(-391/+40),381 bp(-341/+40), 331 bp(-291/+40). Respectively inserted into the polyclonal loci of plasmid PGL4.10, then, transfected HC11, 293 T and goat fetal fibroblasts cells, respectively.We found that the promoter of BLG gene had tissue specificity and the activity of 431 bp(-391/+40) is higher than others’.(3) In order to study the role of introns in gene expression, we used pEGFP-C1 as the skeleton of the carrier, and removed its promoter CMV through the enzyme AseⅠ and NheⅠ, then replaced with the promoter of BLG 431 bp(-391/+40). And then used the intron 1,intron 2 and 3 ˊ UTR as regulatory elements to construct the vectors pE-BLGU11,pE-BLGIN1U11 and pE-BLGINU. Next, the results of quantitative PCR and Western blot experiment supported the conclusion that the introns of BLG gene can enhance the expression ofGFP on the level of mRNA transcription and protein translation, especially the first intron.In summary, this study cloned and analyzed the promoter sequences of β-lactoglobulin gene, and identified the optimal promoter sequence of BLG by gradient cut out. With vector pEGFP- C1, the role of introns of β-lactoglobulin gene in gene expression was preliminary confirmed. We succeed to construct the mammary gland specific expression vector using the regulatory elements cloning from the of goat β-lactoglobulin gene, followed by the further improving the expression level of exogenous gene and producing mammary gland bioreactor.更多还原