乳腺生物反应器特异调控序列的构建

目的构建可以指导外源基因在动物乳腺组织特异表达的调控序列———牛α-S1酪蛋白基因调控序列。方法提取新生小牛肝组织的DNA,应用PCR分段扩增牛α-S1酪蛋白基因调控序列,经琼脂糖凝胶电泳鉴定后测序,再将各基因片段连接成完整的序列。并将外源基因ALB亚克隆入该调控序列内,转染C127细胞,经免疫组化法检测其表达情况。结果乳腺生物反应器特异调控序列———牛α-S1酪蛋白基因调控序列,包括其1.8kb上游序列、第一外显子、第一内含子、包含翻译起始位点的第二外显子和部分第二内含子,及其最后一个内含子的大部分、最后一个不翻译的外显子(含多聚A信号)及侧翼区全长5.8kb。位于该调控序列内的ALB高效表达。结论成功构建乳腺生物反应器特异调控序列———牛α-S1酪蛋白基因调控序列,此调控序列对外源基因的表达有正调控作用。

Construction of Specific Regulating Sequence of Mammary Gland Bioreactor

Objective To construct bovine α-S1 casein gene regulating sequence guiding the specific expression of exogenous gene in animal mammary gland tissue.Methods Extract DNA from the liver tissue of newborn calf for fractional amplification of bovine α-S1 casein gene regulating sequence by PCR.Identify the amplified product by agarose gel electrophoresis and sequencing,then ligate the fragments into a intact one.Subclone the exogenous gene ALB into the regulating sequence for transfection of C127 cells.Identify the expressed product by immunohistochemical method.Results The bovine α-S1 casein gene regulating sequence,consisting of 1.8 kb upstream sequence,exon 1,intron 1,the exon 2 with translation initiation site,a part of intron 2,a large part of the last intron,the last exon(containing poly A signal) which could not be translated and the whole flanking region at a length of 5.8 kb,was constructed.The exogenous gene ALB was highly expressed in the regulating sequence.Conclusion The bovine α-S1 casein gene regulating sequence positively regulating the expression of exogenous gene in animal mammary gland bioreactor was successfully constructed.