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

Gene Sequence Analysis,Protein Structure Prediction and Subcellular Localization of MGF 360-9L from African Swine Fever Virus

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。

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 primary,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 mainlyα-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.