稳定表达绵羊肺腺瘤病毒表面蛋白A549细胞系的建立

Construction of an A549 cell line expressing the surface protein of Jaagsiekte retrovirus

为研究绵羊肺腺瘤病病毒(JSRV)表面蛋白(SU)的致瘤机制,本研究构建稳定表达SU的羊肺细胞A549细胞系。采用PCR方法从含su基因的pGEX-4T-1-SU重组质粒中扩增SU编码序列,将其克隆至真核表达载体pcDNA3.1(+)中,转染A549细胞。通过G418筛选,对转染阳性细胞进行纯化,获得稳定表达JSRVSU蛋白的A549细胞系。以间接免疫荧光及westernblot鉴定SU的表达状况,并运用共聚焦显微镜确认SU蛋白的亚细胞定位。结果表明,重组蛋白SU在A549细胞中有效表达,而且主要分布于细胞质中。该细胞系的建立为SU生物学功能的体外研究提供了平台。

The surface protein （SU） of Jaagsiekte retrovirus （JSRV） is considered closely related to the formation of sheep pulmonary adenomatosis induced with JSRV. To facilitate investigations on SU for its role in transformating target cells, an A549 cell line that stably expresses the JSRV SU was developed in this study. The coding region of the su gene was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.1（＋）. The resulting recombinant plasmid was transfected into the A549 cells, and a transfected cells stably expressing SU, named as A549-SU, was screened and colony-purified under the drug selection of G418. The expression of SU was confirmed by indirect immunofluorescence assay and western blot. Results indicated that the recombinant SU was correctly and effectively expressed in A549-SU and was mostly located in the cytoplasm. This SU-expressing cell line provides a useful in vitro platform for studying biological functions of the JSRV SU.