携带BARF1基因的重组巨细胞病毒的构建

Construction of the recombinant cytomegalovirus carrying BARF1 gene

目的依据细菌人工染色体(Bacterial artificial chromosome,BAC)能够克隆大段DNA病毒的特点,通过galk为基础的同源重组,将EB病毒编码BARF1基因插入巨细胞病毒(cytomegalovirus,CMV),构建重组病毒。方法 PCR扩增带有巨细胞病毒UL57基因左右同源臂的galk及BARF1基因片段,经过电转化,进行同源重组,经含有galk培养基及脱galk培养基筛选,获得带有BARF1的巨细胞病毒细菌人工染色体克隆,提取质粒,转染到ARPE-19细胞,观察带有BARF1基因的巨细胞病毒对ARPE-19细胞的影响。结果感染带有BARF1基因的巨细胞病毒的ARPE-19细胞形态由原来的长梭形变为变圆、肿胀、胞浆颗粒增多,并且细胞生长失去接触抑制,出现重叠生长现象。结论建立了携带BARF1基因的重组巨细胞病毒;同时表明利用细菌人工染色体,可方便地对病毒基因组进行准确操作。

The purpose of this study is to establish recombinant cytomegalovirus carrying BARF1 gene, which based on characteristics of bacterial artificial chromosome can clone a large segment DNA virus through homologous recombination on ba- sis of galk. Galk and BARF1 gene fragment with 50 bp homologous arms of cytomegalovirus UL57 was amplified by PCR, re- spectively. The clones of recombinant cytomegalovirus carrying BARF1 gene was obtained after electroporation through homol- ogous recombination and selection in medium containing galk and replacing galk for BARF1 gene in 2-deoxy-galactose medium. The plasmids of recombinant cytomegalovirus bacterial artificial chromosome containing BARF1 were transfected into ARPE-19 cells after the plasmids extracted from clones. The morphology of ceils infected recombinant virus was changed from the origi- nal long fusiform to round, swelling, and full of cytoplasm particles. The ceils of infected recombinant virus grow overlapping and lost contact inhibition. These results indicated that recombinant cytomegalovirus carrying BARF1 gene was established, and the virus genome can be operated accurately using bacterial artificial chromosomes.