摘要
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。
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.
出处
《中国生物制品学杂志》
CAS
CSCD
2006年第3期275-277,281,共4页
Chinese Journal of Biologicals
基金
深圳市科技计划项目(200104118).
