摘要
旨在通过预测分析和亚细胞定位研究非洲猪瘟病毒(ASFV)D1133L基因结构、亚细胞定位与功能间的关系。作者使用Mega6.0软件绘制了7株不同基因型的D1133L解旋酶和ASFV编码的其他5个解旋酶基因的系统进化树,使用EXPASY、PRABI和SWISS-MODEL软件预测了该基因所编码蛋白序列的二、三级结构;根据GenBank公布的ASFV序列(LR743116.1),合成D1133L基因并构建其重组真核表达质粒pCMV-D1133L,蛋白免疫印迹(Western blot,WB)验证该基因表达后,将重组质粒转染至PK-15细胞,应用间接免疫荧光试验(indirect immunofluorescence assay,IFA)研究了该蛋白的亚细胞定位。结果表明,通过ASFV解旋酶的基因系统进化树,发现5种解旋酶基因均相对保守。D1133L基因全长3402 bp,表达的蛋白为124.63 ku,G+C含量为6.1%,A+T含量为10.6%;二级结构分析表明α螺旋占48.90%,扩展链占13.50%,无规卷曲为33.27%;三级结构分析表明六自由度结构(six degree of freedom,QMQE)为0.20,覆盖率84%,且预测以α螺旋为主的高级结构;通过LOCSVMPSI分析预测,显示D1133L蛋白定位于细胞核内与细胞质内的概率是相同的。WB结果显示pCMV-D1133L质粒正常表达,IFA试验证明ASFV编码的解旋酶D1133L同时分布于细胞核和细胞质。本研究通过对D1133L基因序列、结构功能预测,并通过IFA验证了D1133L亚细胞分布,为进一步揭示D1133L基因功能奠定了一定基础。
The relationships between the structure,subcellular localization and function of D1133L gene of African swine fever virus(ASFV)were explored by predictive analysis and subcellular localization.Mega6.0 software was used to make phylogenetic trees of five helicase genes encoded by ASFV,and Swiss-Model software was used to predict the secondary and tertiary structures of GenBank D1133L gene;according to the ASFV(LR743116.1)sequence published in GenBank(LR743116.1),the D1133L gene was synthesized and its recombinant eukaryotic expression plasmid pCMV-D1133L was constructed.After expression,the gene was verified by Western blot(WB),the recombinant plasmid was transfected into PK-15 cells,and the subcellular localization of the protein was observed with indirect immunofluorescence assay(IFA).The results showed that the 5 ASFV helicases were relatively conservative.The total length of D1133L gene was 3402 bp,G+C content was 6.1%,and A+T content was 10.6%;the expressed protein was 124.36 ku,and the secondary structure indicated that helix accounted for 48.90%,the extended chain accounted for 13.50%,random curl accounted for 33.27%;the tertiary structure indicated that QMQE was 0.20,and the coverage rate was 84%,it has a helix-dominated advanced structure;using LOCSVMPSI analysis and prediction,the probability of D1133L protein localization in the nucleus is the same as that in the cytoplasm.The WB results verified the expression of pCMV-D1133L,and the result of IFA proves that the helicase D1133L encoded by ASFV was localized in the nucleus and cytoplasm at the same time.These results of this study have accumulated data for further study of D1133L protein inhibiting immune response and clarifying the pathogenic mechanism of helicase proteins of ASFV.
作者
侯景
申超超
张大俊
杨博
史喜绢
张婷
崔卉梅
袁兴国
赵登率
陈学辉
张克山
郑海学
刘湘涛
HOU Jing;SHEN Caocao;ZHANG Dajun;YANG Bo;SHI Xijuan;ZHANG Ting;CUI Huimei;YUAN Xingguo;ZHAO Dengshuai;CHEN Xuehui;ZHANG Keshan;ZHENG Haixue;LIU Xiangtao(State Key Laboratory of Pathogenic Biology of Livestock Diseases, Key Laboratory of Animal Virology of the Ministry of Agriculture and Rural Affairs, National Foot-and-Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2021年第7期1953-1962,共10页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
甘肃省科技重大专项-农业领域(20ZD7 NA006)
国家自然科学基金联合项目(31941002)
国家重点研发计划(2018YFC0840400)。
关键词
非洲猪瘟病毒
解旋酶
D1133L基因
序列分析
结构预测
亚细胞定位
African swine fever virus
helicase
D1133L genes
sequence analysis
structural prediction
subcellular localization
