狂犬病病毒糖蛋白哺乳动物细胞稳定表达细胞系的建立

Establishment of Transformed Mammalian Cell Lines Stably Expressing the Rabies Virus Glycoprotein

试验首先根据GenBank上所发表的狂犬病病毒CVS-24株糖蛋白基因序列,设计并合成一对特异性引物,通过反转录-聚合酶链式反应(RT-PCR),获得糖蛋白全长cDNA,连接在pMD18-T载体上,测序证明克隆的正确性后,将其插入真核表达载体pIRES1neo,构建了糖蛋白单一表达载体pICG,表达质粒通过脂质体转染BHK-21细胞,在G418抗性压力下出现细胞克隆,通过PCR检测,确定启动子与糖蛋白基因在细胞基因组中共同整合。借助W estern blot检测,证明所表达的糖蛋白与狂犬病抗血清有特异的反应性。采用间接ELISA法,筛选出3株高效表达糖蛋白的细胞株,分别命名为ICG1、ICG2、ICG3。

The glycoprotein of rabies virus strain CVS-24 was amplified by reverse transcription-polymerase chain reaction and cloned it and its fusion gene with green fluorescent protein into eukaryotic expression vector pIRESlneo. The resultant plasmid pICG was transfeeted into BHK-21 cell line by liposome transfeetion. After screening with G418, CA18-resistant BHK-21 colonies were obtained and amplified. With Western blot analysis, there appeared a reaction band between the specific antibody and the expressed protein, which indicated that the protein has been expressed. By screening with indirect ELISA, three cell clones of expression vector with high level expression were established and used for further identification, named respectively ICG1, ICG2, ICG3.