中国动物传染病学报
中國動物傳染病學報
중국동물전염병학보
CHINESE JOURNAL OF VETERINARY PARASITOLOGY
2014年
2期
27-31
,共5页
郭亦非%郑浩%郑旭晨%童武%刘飞%梁超%单同领%于海%童光志
郭亦非%鄭浩%鄭旭晨%童武%劉飛%樑超%單同領%于海%童光誌
곽역비%정호%정욱신%동무%류비%량초%단동령%우해%동광지
乙型脑炎病毒%NS1'%突变%毒力
乙型腦炎病毒%NS1'%突變%毒力
을형뇌염병독%NS1'%돌변%독력
Japanese encephalitis virus%NS1'%mutation%virulence
通过分析乙型脑炎病毒（Japanese encephalitis virus，JEV）基因组中核糖体移码序列，在对NS2A基因实施同义突变基础上，设计2组引物，通过PCR突变方法，依次删除移码后NS1'基因开放阅读框的终止密码子，并将突变片段替换JEV感染性克隆pJEHEN中相应片段，获得了2株突变全长克隆。经体外转录合成RNA，再以RNA转染细胞，拯救出两株突变病毒vJET1和vJET2。通过RT-PCR扩增突变病毒中含突变位点的基因组片段，测序证实了vJET1基因组3690位核苷酸存在由T向A的突变， vJET2基因组3690位和3819位核苷酸均存在由T向A的突变。将vJET1和vJET2感染Vero细胞后，Western blot检测显示，两株突变病毒表达的NS1'蛋白均出现延长现象。生长曲线显示，与亲本病毒vTHen相比，突变病毒增殖速度均出现下降。动物实验显示，与vTHen相比，vJET1和vJET2对小鼠的神经毒力与神经侵袭力也出现下降。上述结果表明，NS1'编码框的延伸降低病毒在体外培养细胞上的增值速度，也减弱了病毒对小鼠的毒力。
通過分析乙型腦炎病毒（Japanese encephalitis virus，JEV）基因組中覈糖體移碼序列，在對NS2A基因實施同義突變基礎上，設計2組引物，通過PCR突變方法，依次刪除移碼後NS1'基因開放閱讀框的終止密碼子，併將突變片段替換JEV感染性剋隆pJEHEN中相應片段，穫得瞭2株突變全長剋隆。經體外轉錄閤成RNA，再以RNA轉染細胞，拯救齣兩株突變病毒vJET1和vJET2。通過RT-PCR擴增突變病毒中含突變位點的基因組片段，測序證實瞭vJET1基因組3690位覈苷痠存在由T嚮A的突變， vJET2基因組3690位和3819位覈苷痠均存在由T嚮A的突變。將vJET1和vJET2感染Vero細胞後，Western blot檢測顯示，兩株突變病毒錶達的NS1'蛋白均齣現延長現象。生長麯線顯示，與親本病毒vTHen相比，突變病毒增殖速度均齣現下降。動物實驗顯示，與vTHen相比，vJET1和vJET2對小鼠的神經毒力與神經侵襲力也齣現下降。上述結果錶明，NS1'編碼框的延伸降低病毒在體外培養細胞上的增值速度，也減弱瞭病毒對小鼠的毒力。
통과분석을형뇌염병독（Japanese encephalitis virus，JEV）기인조중핵당체이마서렬，재대NS2A기인실시동의돌변기출상，설계2조인물，통과PCR돌변방법，의차산제이마후NS1'기인개방열독광적종지밀마자，병장돌변편단체환JEV감염성극륭pJEHEN중상응편단，획득료2주돌변전장극륭。경체외전록합성RNA，재이RNA전염세포，증구출량주돌변병독vJET1화vJET2。통과RT-PCR확증돌변병독중함돌변위점적기인조편단，측서증실료vJET1기인조3690위핵감산존재유T향A적돌변， vJET2기인조3690위화3819위핵감산균존재유T향A적돌변。장vJET1화vJET2감염Vero세포후，Western blot검측현시，량주돌변병독표체적NS1'단백균출현연장현상。생장곡선현시，여친본병독vTHen상비，돌변병독증식속도균출현하강。동물실험현시，여vTHen상비，vJET1화vJET2대소서적신경독력여신경침습력야출현하강。상술결과표명，NS1'편마광적연신강저병독재체외배양세포상적증치속도，야감약료병독대소서적독력。
To study effect of elongated NS1' protein on virulence of Japanese encephalitis virus, two pairs of primers were designed to eliminate stop codons in NS1' gene without amino acid substitutions in NS2A protein. Two full-length mutant clones pJEHENT1 and pJEHENT2 were constructed from the infectious clone of JEV pJEHEN using site-directed mutation of PCR. Sequencing of the rescued viruses vJET1 and vJET2 revealed a T-A mutation at nt 3690 in vJET1 and the same T-A mutations at nt 3690 and nt 3819 in vJET2. Both vJET1 and vJET2 expressed extended NS1' proteins in Vero cells. The growth curves showed that the mutant viruses vJET1 and vJET2 propagated slower than their parent virus vTHEN. Additionally, reduction of neurovirulence and neuroinvasiveness of vJET1 and vJET2 was noted in mouse pathogenecity test. These results indicated that the elongated NS1' protein slowed down growth of the mutant viruses in Vero cells and decreased their virulence to mice.
