小鼠β-actin基因的RT-PCR克隆与真核表达  

Cloning of Mouse β-actin Gene with RT-PCR and Its Eukaryotic Expression

作　　者：王丹丹[1] 李冬颖[1] 

机构地区：[1]辽东学院农学院,辽宁丹东118003

出　　处：《辽东学院学报（自然科学版）》2016年第1期16-20,共5页Journal of Eastern Liaoning University:Natural Science Edition

摘　　要：以Trizol-异丙醇法提取小鼠肝脏总RNA,根据NCBI数据库中小鼠β-actin基因序列设计引物,采用RT-PCR技术扩增小鼠β-actin基因编码区并测序;以pcDNA3.1-flag为载体,构建β-actin基因真核表达质粒,继而将其重组产物转染于HeLa细胞,采用Western blot印迹法鉴定重组蛋白pcDNA3.1-flag/β-actin的表达水平。结果表明：克隆获得的289 bp片段与NCBI数据库中β-actin基因序列完全吻合,并且成功构建的pcDNA3.1-flag/β-actin真核基因表达载体,其重组质粒可在HeLa细胞中有效表达约43×10^3KD融合蛋白。Total RNA was extracted from mouse livers by the trizol-isopropanol method. The primers were designed and synthesized according to the β-actin gene sequence of mouse in NCBI database. The β-actin gene coding sequence of mouse was amplified and sequenced with RT-PCR. Furthermore,with pcDNA3. 1-flag as the carrier,the eukaryotic expression plasmid was built and its recombinant products were transfect to He La cell. The expression level of recombinant protein pcDNA3. 1-flag / β-actin was appraised with Western blot method. The results showed that the cloned 289 fragment was identical with the β-actin gene sequence in NCBI database. Besides,the recombinant plasmid of built by pcDNA3. 1-flag eukaryotic expression carrier can effectively present 43× 10^3 KD fusion protein.

关 键 词：小鼠 Β-ACTIN基因 RT-PCR 克隆 真核表达 

分 类 号：Q976-33[生物学]