【作者】 陈刚；

【导师】 孙怀昌；

【作者基本信息】 扬州大学 ， 动物学， 2002， 硕士

【摘要】 为了构建具有自主知识产权的山羊乳腺特异性表达载体，利用高保真聚合酶链式反应(PCR)从中国奶山羊基因组中扩增出6个β-乳球蛋白(BLG)基因片段和1个β-酪蛋白(β-casein)基因片段。序列测定结果证明，所获基因片段的DNA序列与已发表的山羊及绵羊相应基因区域具有很高的同源性，5-端调控区中主要的乳腺特异表达调控元件完全相同，不仅说明此两个基因在山羊、奶山羊和绵羊之间是高度保守的，而且说明所用的DNA聚合酶具有很高的保真性，所扩增的基因片段可用于乳腺特异表达载体的构建。将上述基因片段进行组合，构建成一系列乳腺特异性表达载体。将lacZ报告基因分别克隆入自行构建的p205C3载体和从国外引进的pCX及pHC载体，将所获得的重组载体分别用脂质体介导法转染奶山羊乳腺上皮细胞系，经泌乳激素诱导后用X-gal进行酶组化染色，结果表明自行构建的载体驱动报告基因的表达水平高于从国外引进的、已知具有高表达性能的载体。上述重组载体在COS—1细胞和山羊皮肤成纤维细胞不能表达相应的报告基因，表明其具有较好的组织特异性。根据上述实验结果可以得出结论，本研究构建的乳腺特异性表达载体能用于动物乳腺生物反应器的研制。更多还原

【Abstract】 To construct mammary gland-specific expression vectors, 6 β-lactoglobulin (BLG) gene fragments and 1 β-casein gene fragment of expected sizes were amplified from Chinese dairy goat genomic DNA using high fidelity long template PCR system. Partial sequence analyses showed that all 7 gene fragments were more than 99% identical to that of goat and sheep, especially the key regulatory elements in the 5’-end regulatory region of the BLG gene with 100% identity among the three species. This indicates the high fidelity of the DNA polymerase used and the usability of the cloned gene fragments for construction of mammary gland-specific expression vectors. A series of mammary gland-specific expression vectors were constructed by combination of the gene fragments, one of which, known as p205C3, was studied in more detail. The lacZ reporter gene was cloned into the self-constructed vector p205C3 and into two imported vectors pCX and pHC, high expression abilities of which have been demonstrated in both transgenic mice and larger animals. The three recombinant vectors were transfected into goat mammary epithelial cells by using liposome-mediated method, and the transfected cells were stained with a solution containing X-gal following induction with insulin, prolactin and corticosterone. The results showed that the reporter gene was efficiently expressed in the cells transfected with all the three recombinant vectors, but the expression levels in the cells transfected with the self-constructed vector were higher than that with the two imported vectors. The three vectors could not direct the expression of the reporter gene in both COS-1 cells and goat skin fibroblasts. These results suggest that the regulatory sequences of the BLG and p-casein gene cloned from the Chinese dairy goat genomic DNA using high fidelity PCR were usable and the self-constructed goat mammary gland-specific expression vector could be used for generation of mammary gland bioreactors.更多还原