非洲猪瘟病毒解旋酶D1133L基因序列分析、蛋白结构预测及亚细胞定位

doi:10.11843/j.issn.0366-6964.2021.07.017

Abstract

Abstract: 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 3 402 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.

Key words: African swine fever virus, helicase, D1133L genes, sequence analysis, structural prediction, subcellular localization

CLC Number:

S852.659.1

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. Gene Sequence Analysis, Protein Structure Prediction and Subcellular Localization of African Swine Fever Virus Helicase D1133L[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7): 1953-1962.

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Gene Sequence Analysis, Protein Structure Prediction and Subcellular Localization of African Swine Fever Virus Helicase D1133L

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