非洲猪瘟病毒MGF505-5R基因分子特征及蛋白表达载体构建  

Molecular Characterization and Protein Expression Vector Construction of African swine fever virus MGF505-5R Gene

作　　者：沈兆基 谢春嫡 沙嘎那日·吉日木图 郭肖蓉 周荣[2] 李奎[2] SHEN Zhao-Ji;XIE Chun-Di;SHAGAINAR Jurmt;GUO Xiao-Rong;ZHOU Rong;LI Kui(School of Life Science and Engineering,Foshan University/Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding,Foshan 528225,China;Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]佛山科学技术学院,生命科学与工程学院/广东省动物分子设计与精准育种重点实验室,佛山528225 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《农业生物技术学报》2023年第5期1100-1110,共11页Journal of Agricultural Biotechnology

基　　金：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。

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�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(IPECJ2)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 c

关 键 词：猪 非洲猪瘟病毒 蛋白结构 真核表达载体 

分 类 号：S813.1[农业科学—畜牧学]