启动子和PolyA序列对水牛乳腺特异性表达载体表达效率的影响  

Impact of Promoters and Poly A Sequence on Expression Efficiency of Buffalo Mammary Gland Specific Expressing Vectors

作　　者：罗婵[1] 任艳萍[1] 屈春凤 李海洋[1] 李湘萍[1] 石德顺[1] 

机构地区：[1]广西大学动物繁殖研究所,亚热带农业生物资源保护与利用国家重点实验室,广西南宁530004

出　　处：《中国畜牧杂志》2015年第9期62-67,共6页Chinese Journal of Animal Science

基　　金：国家“863”重大专项(2011AA100607);广西亚热带生物资源保护利用重点实验室开放课题(SB1002)

摘　　要：为了获得能有效表达促乳素的乳腺特异性表达载体,本研究系统比较了不同长度的启动子和有/无PolyA序列对乳腺特异性表达载体表达标记基因绿色荧光蛋白(GFP)和目的基因促乳素(PRL)的影响。将不同载体瞬时转染Bcap37细胞后,分别用实时定量PCR和流式细胞仪检测PRL和GFP的表达水平。结果表明:长度为5.2 kb的β-酪蛋白(Beta casein,BCN)启动子启动PRL的表达量显著高于长度为3.8 kb启动子,前者的表达量约为后者的6倍;在BCN启动子长度均为5.2 kb情况下,添加PolyA的载体p5.2BCN-PRL-PolyA-CMV-GFP-Neo组PRL的相对表达量为8.17,p5.2BCN-PRL-PolyA-CMV-GFP组PRL的相对表达量为17.84,均显著高于p5.2BCNPRL-CMV-GFP-Neo组,可见添加PolyA后,PRL的表达量均显著增加;进一步比较发现,含PolyA的载体中GFP的表达水平也显著高于不含PolyA的载体。对比以上结果发现,添加PolyA能够有效解除启动子之间的转录干扰,使标记基因的启动子CMV和目的基因的启动子BCN均能顺利启动基因表达。经过优化的载体启动目的基因PRL表达的效率更高,可应用于下一步的研究工作中。To investigate the impact of promoters and PolyA sequence on the expression of exogenous genes in Bcap37 cells, two different length of Beta casein （BCN） promoters and PolyA sequences were used in the constitute of buffalo mammary gland specific expressing vectors. Real-time PCR and flow cytometry analysis were used to detect the expression levels of marker gene green fluorescent protein （GFP） and target gene prolactin （PRL） in Bcap37 cells transfected with different vectors. The result showed that the expression level of PRL was significant higher in the cells transfected with vector contained a 5.2 Kb length of BCN promoter than vector contained 3.8 Kb length of BCN promoter. It indicated that the expression of exogenous was positively correlated to the length of the promoters. Moreover, both the expression of GFP and PRL were improved remarkably when inserting PolyA sequence between promoter CMV and BCN, the results showed that the inserted PolyA sequences could eliminate the transcription inhibition inducing by promoter CMV and BCN. The modified vectors which driven the expression of PRL more effectively can be utilized in the subsequent work.

关 键 词：启动子 PolyA序列 促乳素 绿色荧光蛋白 基因表达 水牛 

分 类 号：S823.2[农业科学—畜牧学]