不同长度poly(C)对口蹄疫病毒基因工程毒的毒力影响  

Virulence comparison of genetic engineering virus containing different length poly(C) tract of foot-and-mouth disease virus

作　　者：白兴文[1] 李平花[1] 孙普[1] 李永亮[1] 包慧芳[1] 卢曾军[1] 曹轶梅[1] 郭建宏[1] 刘在新[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,国家口蹄疫参考实验室,农业部畜禽病毒学重点开放实验室,兰州730046

出　　处：《微生物学报》2008年第12期1654-1658,共5页Acta Microbiologica Sinica

基　　金：国家“973项目”(2005CB23201);“十一五”国家科技支撑计划项目(2006BAD06A03)~~

摘　　要：【目的】研究口蹄疫病毒(foot-and-mouth disease virus,FMDV)poly(C)区段的序列长度与FMDV毒力之间的关系。【方法】首先利用NheⅠ/NotⅠ线化FMDV pGEM-XJ/AKT/69全长重组质粒,制备体外转录本,借助Lipofectamine 2000转染BHK-21细胞,获得基因工程毒。随后,在细胞传代过程中,收取不同代数病毒培养液,提取总RNA进行poly(C)的长度测定,并进行乳鼠致病性试验和BHK-21细胞TCID_(50)的测定。【结果】我们发现,基因工程毒在BHK-21上连续传代至第6代时,可见明显的致细胞病变效应(cytopathic effect,CPE);在经过一代乳鼠接种之后再重新适应到BHK-21细胞的过程中,poly(C)区段出现了缩短现象;乳鼠致病性试验和BHK-21细胞TCID_(50)测定结果表明,尽管基因工程毒与母本毒相比毒力偏低,但含有不同长度poly(C)的基因工程毒之间并无显著差异。【结论】poly(C)区段的序列长度在12～17个C之间改变对FMDV基因工程毒的毒力无明显的影响。[Objective] Study the association of the length of poly（C） tract with virulence of foot-and-mouth disease virus. [Methods] The recombinant plasmids pGEM-XJ/AKT/69 containing the full-length cDNA of FMDV were linearized by Nhe Ⅰ/Not Ⅰ and transcribed by T7 RNA polymerase. The in vitro transcripts were transfected into BHK-21 cells using Lipofectamine 2000 reagent. Then, the genetic engineering virus was rescued from BHK-21 cells. The poly（C） tracts were sequenced at different passages of rescued virus, and the pathogenicities were evaluated with 3-day-old mice and BHK-21 cells by detection of LD50 and TCID50. [Results] After six passages in BHK-21 cells, cytopathic effect was observed by microscopy. The rescued virus was rejuvenated once in unweaned mice, and then came back to cell passage. We found that the poly（C） tract of rescued virus was shortened when the virus was passaged twice in BHK-21 cells. Although the data of LD50 in mice and TCID50 in BHK-21 cells showed that the virulence and infectivity of genetic engineering viruses were lower than its parental virus, no significant difference was observed between the genetic engineering viruses with the different length poly（C） tract. [Conclusion] The length of poly（C） tract ranged from 12 to 17 nucleotides did not cause significant influence on the virulence and infectivity of genetic engineering virus.

关 键 词：口蹄疫病毒 基因工程毒 poly(C) 毒力 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]