【作者】 任艳萍； 黄章虎； 刘庆友； 石德顺；

【Author】 REN Yan-ping,HUANG Zhang-hu,LIU Qing-you,SHI De-shun~* (College of Animal Science and Technology,Guangxi University,Nanning 530005,China)

【机构】 广西大学动物科学技术学院；

【摘要】 催乳素(Prolactin,PRL)可以刺激乳腺导管系统的发育,启动并维持泌乳,与产奶量密切相关。本文构建了水牛乳腺特异表达PRL基因的载体,并在细胞水平进行了表达检测。参考已公布的奶牛和部分水牛PRL序列,设计特异性扩增引物,提取成年母水牛垂体总RNA,经RT-PCR扩增获得长804bp的水牛PRL全长CDS序列,克隆和测序结果显示与奶牛序列相似性达98.6%。应用双酶切定向克隆技术,将PRL基因片段、β酪蛋白的5’和3’调控序列片段、以及标记基因EGFP和NEO片段,经酶切-胶回收-连接-转化-筛选等常规基因克隆步骤,建成了乳腺特异性表达PRL载体pBCN-PRL-polyA-cmv-EGFP-sv40-NEO,经PCR、酶切和测序检测所构建的载体与预期设计符合。将载体应用脂质体法转染人乳腺癌细胞,24h后可观察到EGFP的表达,提取转染细胞总RNA,经RT-PCR可扩增到PRL基因的特异条带,经Western Blot检测可观察到2.2kD的特异性条带,而对照组RT-PCR和Western blot结果均未发现PRL基因条带,初步证明构建的载体能够在乳腺表达PRL基因。应用此载体研制转基因水牛,预期PRL基因仅在成年母水牛乳腺中特异表达,促进并维持泌乳,提高水牛产奶量,与此同时避免了选用通用启动子时,全身过量表达PRL,而引发类似高催乳素血症的症状,为获得高繁殖力转基因水牛奠定了工作基础。更多还原

【Abstract】 Prolactin(PRL) gene plays an important role in promoting growth of mammary duct and acinus system growth,starting and maintaining milk secrete,and is closely related to milk yield.In this study,a mammary glands specific expression PRL transgenic vector was constructed and detected its expressing in cellular level.Referencing the released cattle PRL sequence and part of buffalo PRL sequence,a pair of primers was designed,which includes the whole CDS sequence of buffalo PRL.With total RNA isolated from Hyperprolactinemia of adult female buffalo as template,an 804bp fragment was amplified by RT-PCR.The fragment was cloned and sequenced,and the sequence was 98.6%similar to that of cattle PRL gene.Using double-enzyme directional cloning technology,with five fragments including PRL CDS,3’ and 5’ regulator sequence of beta-casein,marker gene(EGFP) and antibiotics resistance gene(NEO) as material,after several cycle steps(specific restriction enzyme digestion,gel extraction purification,ligation,transformation and picking the positive plasmid),a mammary glands specific expression PRL vector p-BCN-PRL-polyA-cmv-EGFP-sv40-NEO was constructed at last.The vector was assayed by PCR,restriction enzyme and sequencing,and all results accorded with the primary design.Then the vector was transfected into BcaP cells with lipofectamine,and green fluorescence protein was observed in part of transfected cells at 24h. With total RNA of these transfected cells as template,the expected band was observed via RT-PCR technique. With Western blot analysis,2.2kD PRL protein band was assayed in total proteins of these transfected cells.It is expected that transgenic buffaloes produced by the vector,would specifically express PRL in mammary glands of adult female buffaloes,which would increase milk yield of buffaloes,while the buffalo expressing PRL by general promoter would overexpress PRL and would suffer from Hyperprolactinemia.更多还原