非洲猪瘟病毒MGF 360-9L基因序列分析、蛋白结构预测及亚细胞定位

doi:10.11843/j.issn.0366-6964.2020.020

摘要/Abstract

摘要： 前期研究结果表明，非洲猪瘟病毒（ASFV） MGF 360-9L能显著抑制宿主天然免疫应答，故通过比较、分析ASFV中MGF 360基因序列，进一步研究MGF 360-9L基因结构和功能间的关系。本研究采用生物信息学方法，分析该基因的一级结构并预测该基因的表达蛋白二、三级结构；根据GenBank公布的Georgia 2007/1（FR682468.1）序列，合成MGF 360-9L基因并构建其重组真核表达质粒，Western blot验证该基因表达后，将重组质粒转染至PK-15细胞，经染色后观察其蛋白的亚细胞定位。结果表明，以Ⅱ型Georgia 2007/1基因组中MGF 360-9L基因为参照，其在Ⅱ型ASFV毒株中高度保守，在54株不同基因型毒株间的相似性与ASFV系统进化关系一致，保守序列为3'末端378 bp片段，高级结构以α螺旋为主，核定位序列预测其定位于细胞核内；构建真核表达质粒，成功表达目的蛋白且定位于细胞核内。本研究结果为进一步研究MGF 360-9L基因抑制免疫应答和明确MGF 360基因家族在ASFV的致病机制积累了资料。

关键词: 非洲猪瘟病毒, MGF 360-9L基因, 序列分析, 结构预测, 亚细胞定位

Abstract: Previous research results showed that African swine fever virus (ASFV) MGF360-9L can significantly inhibit the host's innate immune response. Herein we compared and analyzed MGF360-9L gene sequences of ASFV and predicted the relationship between MGF360-9L structure and function. In this study, bioinformatics method was used to analyze and predict its pri-mary, secondary and tertiary structure of the expressed protein. Based on the sequence of Georgia 2007/1 published in GenBank (FR682468.1), the MGF360-9L gene was synthesized and its recombinant eukaryotic expression plasmid was constructed. The recombinant plasmid was transfected into PK-15 cells, then gene expression was identified by Western blotting, and subcellular localization of this protein was observed by indirect immunoinfluscent assay (IFA). Results showed that MGF360-9L gene is highly conserved among type II ASFV strains, Georgia 2007/1 is the representative virus strain. The homology analysis results of 54 different ASFV strains is consistent with its phylogenetic relationships of them, the conserved sequence is a 378 bp fragment at the 3'terminal of MGF360-9L. The advanced structure of this protein was mainly α-helix. Nuclear location sequence (NLS) prediction revealed that it might be located in the nucleus, and is consistent with the results of MGF360-9L IFA in PK-15 cells. This study accumulated significant data for further research on inhibition of innate immune response by ASFV MGF360-9L and pathogenic mechanisms of the MGF360 gene family in ASFV.

Key words: African swine fever virus, MGF 360-9L gene, sequence analysis, structural prediction, subcellular localization

中图分类号:

S852.659.1

申超超, 李国丽, 张大俊, 徐国伟, 侯景, 李丹, 党文, 张克山, 郑海学, 刘湘涛. 非洲猪瘟病毒MGF 360-9L基因序列分析、蛋白结构预测及亚细胞定位[J]. 畜牧兽医学报, 2020, 51(6): 1371-1381.

SHEN Chaochao, LI Guoli, ZHANG Dajun, XU Guowei, HOU Jing, LI Dan, DANG Wen, ZHANG Keshan, ZHENG Haixue, LIU Xiangtao. Gene Sequence Analysis, Protein Structure Prediction and Subcellular Localization of MGF 360-9L from African Swine Fever Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1371-1381.

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