摘要
目的构建登革2型病毒非结构蛋白NS3及其结构域基因的真核表达载体,并鉴定重组蛋白在BHK-21细胞内对登革病毒复制的抑制作用。方法以登革2型病毒新几内亚毒株(NGC DENV-2)基因组RNA为模板,设计引物扩增获得编码NS3及其蛋白酶NS3P和解旋酶NS3H的基因,通过基因重组的方法分别将3段基因片段克隆入真核表达载体pcDNA3.1(+),获得重组表达载体pcDNA3.1-NS3、pcDNA3.1-NS3P和pcDNA3.1-NS3H;经脂质体法分别转染BHK-21细胞后,用RT-PCR、间接免疫荧光和Western印迹鉴定表达的蛋白。结果成功构建pcNS3、pcNS3P和pcNS3H重组质粒,转染进BHK-21细胞48h后,分别检测出相对分子量约为69kDa、50kDa及18kDa的特异蛋白;且转染pcNS3、pcNS3H组均与空白对照组及未转染组间的病毒载量存在着差异(P<0.05);转染pcNS3P组与未转染组及空白对照组的病毒载量无统计学差异(P>0.05)。结论 DENV-2的非结构蛋白NS3能够抑制病毒的复制。
The recombinant eukaryotic plasmid expressing the nonstructural protein NS3 of dengue 2 virus was constructed in this study and the function of inhibiting virus replication of the recombinant proteins in the BHK-21 cells was identified.The gene fragments of NS3,NS3P and NS3H were amplified by PCR from full-length cDNA cloned vector of dengue 2 virus （NGC）,and then were cloned into eukaryotic expression vector pcDNA3.1（＋） to generate the desired recombinant plasmid peNS3,pcNS3H and pcNS3P.They were transiently transinfected into the mammalian BHK-21 cells by lipofectamine 2000,and the expression of recombinant proteins were identified by RT-PCR,Indirect immunofluorescence and Western blot.Results showed that all the recombinant proteins DV2-NS3,DV2-NS3P and DV2-NS3H existed mainly in the cytoplasm,and the proteins of 69 kDa,50 kDa and 18 kDa were detected.The viral load was significant difference （P〈0.05） between pcNS3 or pc-NS3H group and the untransinfected/control group.There was no significant difference （P〉0.05） between pcNS3p and the untransinfected/control group.It shows that the NS3 protein has a role in inhibiting virus replication.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2014年第5期464-468,共5页
Chinese Journal of Zoonoses
基金
国家自然科学基金(No.81271880)资助~~
关键词
登革病毒
非结构蛋白NS3
真核表达
dengue virus
nonstructural protein NS3
eukaryotic expression
