非洲猪瘟病毒 <em>MGF505</em>-<em>5R</em> 基因分子特征及蛋白表达载体构建

doi:10.3969/j.issn.1674-7968.2023.05.019

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摘要非洲猪瘟(African swine fever, ASF)是一种对猪(Sus scrofa)具有毁灭性的疾病,通常引发出血热, 对养猪业危害巨大。多基因家族(multigene families, MGF) 505-5R 位于非洲猪瘟病毒(African swine fever virus, ASFV)基因组右侧可变区域(the right variable region, RVR),能够影响病毒的毒力。本研究旨在分析 MGF505-5R 基因和蛋白的分子特征,构建MGF505-5R基因真核表达载体。根据ASFV参考基因组(GenBank No. MK128995.1)提取 MGF505-5R 基因序列进行生物信息学分析, 并合成 MGF505-5R 基因片段。通过同源重组的方式将 MGF505-5R 基因连接至 pRK5M-C-2×Strep 载体。经 PCR 和测序鉴定后, 将重组质粒 pRK5M-C-2×Strep-MGF505-5R 转染至猪小肠上皮细胞系(porcine intestinal columnar epithelial cells, IPEC-J2)中, 通过免疫荧光和 Western blot 检测 MGF505-5R 的基因表达效果。通过 qPCR 检测过表达 pRK5M-C-2×Strep-MGF505-5R 的 IPEC-J2 细胞中外切体(exosome)8个亚基、Mtr4 外切体 RNA 解旋酶(Mtr4 exosome RNA helicase, MTREX)、末端核苷酸转移酶 4A (terminal nucleotidyltransferase 4A, TENT4A)、锌指 DHHC 型棕榈酰转移酶 16 (zinc finger DHHC-type palmitoyltransferase 16, ZDHHC16)基因的表达。生物信息学分析结果表明,MGF505-5R 基因在系统发育树中分为3个分支,MGF505-5R 蛋白在20、105 和 485 位氨基酸存在正向选择。MGF505-5R 蛋白的二级结构与三级结构相符, 正选择位点均位于无规则卷曲上。PCR 结果显示 MGF505-5R 基因片段成功连接至 pRK5M-C-2×Strep 载体中。免疫荧光和Western blot 检测结果显示, MGF505-5R 蛋白能够在猪 IPEC-J2 细胞中表达。qPCR 结果显示, 过表达pRK5M-C-2×Strep-MGF505-5R 的 IPEC-J2 细胞中外切体组分(exosome component, EXOSC) 1、EXOSC2、EXOSC3、EXOSC4、EXOSC5、EXOSC7、EXOSC8、EXOSC10、TENT4A 和 ZDHHC16 的表达量极显著降低(P<0.01);MTREX 的表达量无显著变化。本研究对 MGF505-5R 分子特征的分析,为该基因的进化研究提供基础; 构建 MGF505-5R 在猪 IPEC-J2 细胞的表达载体, 为 ASFV 与宿主之间的蛋白质相互作用研究提供有效的工具和实验素材。

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关键词 ：猪, 非洲猪瘟病毒, 蛋白结构, 真核表达载体

Abstract：African swine fever (ASF), a devastating disease for pigs (Sus scrofa), usually causes hemorrhagic fever, which bring great economic losses to the pig industry. Multigene families (MGF) 505-5R was in the right variable region (RVR) of the African swine fever virus (ASFV) genome and could influence the virulence of the virus. The work aimed to analyze the molecular characteristics of the MGF505-5R gene and protein and construct a eukaryotic expression vector for the ASFV MGF505-5R gene. The MGF505-5R gene was obtained from the ASFV reference genome in GenBank (GenBank No. MK128995.1) for bioinformatics analysis, and the MGF505-5R gene was synthesized. The MGF505-5R gene was ligated into the pRK5M-C-2×Strep vector by homologous recombination. The recombinant plasmid pRK5M-C-2×Strep-MGF505-5R was transfected into IPEC-J2 cells after being identified by PCR and sequencing, and the expression of the MGF505-5R gene was confirmed by immunofluorescence and Western blot. Transcript levels of exosome 8 subunits, Mtr4 exosome RNA helicase (MTREX), terminal nucleotidyltransferase 4A (TENT4A) and zinc finger DHHC-type palmitoyltransferase 16 (ZDHHC16) in IPEC-J2 cells overexpressing pRK5M-C-2×Strep-MGF505-5R were detected by qPCR. Bioinformatics analysis showed that the MGF505-5R gene was divided into 3 branches in the phylogenetic tree and there was a positive selection at amino acids 20, 105 and 485. The secondary structure of MGF505-5R protein matched the tertiary structure, and the positive selection sites were all located on the irregularly coiling. PCR results showed that the MGF505-5R gene fragment was successfully ligated into the pRK5M-C-2×Strep vector. Immunofluorescence and Western blot assays showed that the pRK5M-C-2×Strep-MGF505-5R vector was successfully expressed in porcine intestinal columnar epithelial cells (IPEC-J2) cells. The qPCR results showed that the expression of exocytotic complex subunits exosome component (EXOSC) 1, EXOSC2, EXOSC3, EXOSC4, EXOSC5, EXOSC7, EXOSC8, EXOSC10, TENT4A and ZDHHC16 was extremely significantly decreased in IPEC-J2 cells overexpressing pRK5M-C-2×Strep-MGF505-5R (P<0.01); no significant change in MTREX expression. The analysis of the molecular characteristics of MGF505-5R in this study provided a basis for the evolutionary study of this gene; the construction of the MGF505-5R expression vector in pig IPEC-J2 cells provides the effective tool and experimental material for the study of protein interactions between ASFV and the host.

Key words： Pigs Africa swine fever virus Protein structure Eukaryotic expression vector

收稿日期: 2022-03-14

ZTFLH:

S813.1

基金资助:中央级公益性科研院所基本科研业务费专项(Y2021XK20); 国家自然科学基金(31972541); 六安市产学研合作重大专项

通讯作者: *zhourong03@caas.cn

引用本文:

沈兆基, 谢春嫡, 沙嘎那日·吉日木图, 郭肖蓉, 周荣, 李奎. 非洲猪瘟病毒 MGF505-5R 基因分子特征及蛋白表达载体构建[J]. 农业生物技术学报, 2023, 31(5): 1100-1110.
SHEN Zhao-Ji, XIE Chun-Di, SHAGAINAR Jurmt, GUO Xiao-Rong, ZHOU Rong, LI Kui. Molecular Characterization and Protein Expression Vector Construction of African swine fever virus MGF505-5R Gene. 农业生物技术学报, 2023, 31(5): 1100-1110.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.05.019 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I5/1100

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