绵羊肺腺瘤病毒内蒙株衣壳蛋白在真核细胞中的表达

Eukaryotic Expression of CA Gene of Jaagsiekte Sheep Retrovirus Inner Mongolian Strain

为构建绵羊肺腺瘤病毒(Jaagsiekte Sheep Retrovirus,JSRV)内蒙株衣壳蛋白(CA)基因真核表达载体,采用含HA标签的引物,通过PCR方法从重组原核表达质粒上扩增CA-HA融合基因片段,将其克隆至真核表达载体上。构建的真核表达质粒通过脂质体法转染293细胞和Hela细胞,用间接免疫荧光和Westhern blotting法检测目的蛋白的表达。结果表明,成功地构建真核表达质粒pcDNA3.1(+)-CA-HA,并转染的293细胞和Hela细胞内观察到特异性荧光和检测到目的基因的表达产物,说明CA基因在真核细胞内获得表达。本试验为建立特异性的诊断试剂盒奠定了基础。

In order to construct CA gene expression vector that directs the synthesis of protein in Eu- karyotic cells, the CA-HA fusion gene was amplified using primers with HA-tag sequence from the recom- binant plasmid pGEX-4T-1-CA and cloned into a plasmid pcDNA3.1（＋）. The recombinant plasmid pcD- NA3. I（＋）-CA-HA was transfected into 293 cells and Hela cells. CA expression was confirmed by indi- rect immunefluorescence and Western blot analysis by using anti-HA monoclonal antibody. Results revealed that the protein was expressed in 293 ceils and Hela cells as expected. The results laid a foundation for the development of genetically engineered diagnostic reagents of JSRV Inner Mongolia strain.