乳腺特异性表达催乳素基因载体的构建与检测  

The Construction and Detection of Nuclear Transfer Vector Specifically Expressing PRL Gene in the Mammary Gland

作　　者：任艳萍[1,2] 雷小灿[1,2] 谢亮亮[1] 罗婵[1] 刘晓华[1] 石德顺[1] 李湘萍[1] 

机构地区：[1]广西大学亚热带农业生物资源保护与利用国家重点实验室,南宁530004 [2]遵义医学院基础医学院,遵义563003

出　　处：《中国细胞生物学学报》2016年第5期519-526,共8页Chinese Journal of Cell Biology

基　　金：国家转基因重大专项(批准号:2014ZX08007-001);国家自然科学基金(批准号:31560632);广西自然科学基金项目(批准号:2014GXNSFAA118084);亚热带农业生物资源保护与利用国家重点实验室开放基金(批准号:KSL-CUSAb-2012-02)资助的课题~~

摘　　要：催乳素(prolactin,PRL)可通过PRL-PRLR-JAK/STAT信号通路促进乳腺发育,启动并维持泌乳。为了探讨调控PRL基因表达对奶水牛产奶量的影响,该研究构建了乳腺特异性表达PRL基因的核移植载体并检测了其有效性。首先,利用RT-PCR方法克隆得到804 bp的水牛PRL基因编码区;而后逐步采用酶切加连接方法,依次将PRL基因、β-酪蛋白(β-casein,BCN)启动子和标记基因插入p IFN-BCNpoly A质粒中,构建得到14.2 Kb的转PRL基因载体。将表达载体瞬时转染人Bcap-37细胞系,经RT-PCR检测发现,目的基因PRL可在该细胞系中表达。将该载体转入水牛胎儿成纤维细胞中,通过核移植法获得了转PRL基因水牛克隆胚胎。该文结果表明,所构建的PRL核移植载体可表达PRL基因,并可用于生产转PRL基因克隆水牛胚胎。Prolactin （PRL） could improve the development of mammary gland, promote and maintain the lactation by PRL-PRLR-JAK/STAT signaling pathway. In order to investigate the influence of the buffalo mike yield regulated by PRL gene expression, the nuclear transfer vector specifically expressing PRL gene in the mammary gland was constructed, and its effect was detected in this study. At first, the 804 bp PRL gene coding region of female buffalo was cloned by RT-PCR method. The 14.2 Kb PRL transgenic vector was constructed by inserting PRL gene, BCN （β-casein） promoter and marker gene into plFN-BCNpolyA plasmid using restriction and ligation methods. The PRL vector was transiently transfected into human Bcap-37 cell line, and the expression of PRL target gene was detectable in the transfected cells. Finally, the vector was transfected into the buffalo fetal fibroblast cells, and the PRL transgenic cloning buffalo embryos were produced by nuclear transfer method. Our results indicated that the constructed vector could express PRL gene, and could be applied for producing PRL transgenic cloning buffalo embryos.

关 键 词：PRL 乳腺特异性表达 载体构建 转基因动物 核移植 

分 类 号：S823[农业科学—畜牧学] Q78[农业科学—畜牧兽医]