摘要
【目的】对苏山猪甘露糖苷酶α2A类成员1(mannosidase alpha class 2A member 1,MAN2A1)基因进行克隆和生物信息学分析,并揭示MAN2A1基因过表达对猪流感病毒(Swine influenza virus,SIV)复制的调控作用。【方法】本研究以苏山猪肺脏cDNA为模板扩增MAN2A1基因编码区序列(CDS),并将目的片段通过T4 DNA连接酶克隆到pMD19-T载体上。将序列与其他物种及不同品种猪进行相似性比对并构建系统发育树;利用生物信息学软件对苏山猪MAN2A1蛋白序列特征进行分析;使用实时荧光定量PCR检测MAN2A1基因在苏山猪中的组织表达谱及MAN2A1基因过表达前后SIV M和NP基因的表达水平;使用AutoDock软件对MAN2A1蛋白与SIV M和NP蛋白进行分子对接预测。【结果】本研究成功克隆了苏山猪MAN2A1基因CDS区序列,其长度为3435 bp。与NCBI数据库公开的猪的MAN2A1基因序列(XM_003123823.6)相比,苏山猪MAN2A1基因CDS区存在6个单核苷酸多态位点(single nucleotide polymorphism sites,SNPs),包含2个错义突变和4个同义突变。不同物种MAN2A1基因系统进化树显示,猪MAN2A1基因与牛和羊亲缘关系最近,与灵长类关系次之,与家禽和斑马鱼亲缘关系最远;不同品种猪MAN2A1基因系统进化树显示,苏山猪MAN2A1基因与中国五指山猪亲缘关系最近,与杜洛克亲缘关系最远。生物信息学分析结果显示,苏山猪MAN2A1蛋白含1144个氨基酸,分子质量为131.42 ku,理论等电点为7.85;二级结构中α-螺旋、β-折叠、无规则卷曲占比分别为33.65%、15.73%和50.61%;三级结构全局模型质量评估GMQE评分为0.9,质量较好;苏山猪MAN2A1蛋白含有3个结构域(第168-498、289-503和649-1140位氨基酸),主要定位在细胞质内的高尔基体中,可能与MAN1A1、FUT8和COPB2等蛋白存在相互作用。组织表达谱检测结果表明,苏山猪MAN2A1基因在肝脏和脾脏中的表达水平显著高于其他组织(P<0.05),在心脏和肌肉中的表达水平显著低于其他组织(P<0.05)。本研究成功构建了MAN2A1基因的过表达载体,与对照组相比,过表达组3D4/21细胞中MAN2A1基因水平显著上调(P<0.05);感染H1N1亚型SIV时,过表达MAN2A1基因导致病毒M和NP基因表达水平显著上调(P<0.05)。分子对接预测结果显示,MAN2A1蛋白与H1N1亚型SIV NP、M1和M2蛋白均可能存在相互作用关系(iptm+ptm>0.9),表明MAN2A1蛋白可能直接作用于NP和M蛋白进而调控H1N1亚型SIV的复制。【结论】本研究成功克隆了苏山猪MAN2A1基因CDS区序列,并发现苏山猪MAN2A1基因过表达促进了H1N1亚型SIV在3D4/21细胞中的复制,上述研究结果为进一步研究MAN2A1基因的功能和分子机制提供了理论依据。
【Objective】This study aimed to clone and perform biological analysis of mannosidase alpha class 2A member 1(MAN2A1)gene in Sushan pigs,as well as reveal the regulation of MAN2A1 gene overexpression on the replication of Swine influenza virus(SIV).【Method】The complete CDS region sequence of MAN2A1 gene was amplified using porcine lung cDNA as a template and ligated into the pMD19-T vector with T4 DNA ligase.The similarities of the sequence with other species and different breeds of pigs were compared and phylogenetic trees were constructed.Bioinformatics software was used to predict the MAN2A1 protein sequence of Sushan pigs.Real-time quantitative PCR was used to detect the tissue expression profile of MAN2A1 gene in Sushan pigs,as well as the expression levels of SIV M and NP genes before and after overexpression of MAN2A1 gene.AutoDock software was used to predict molecular docking between MAN2A1 protein and SIV M and NP proteins.【Result】The results showed that the CDS region sequence of porcine MAN2A1 gene was successfully cloned,which was 3435 bp in length.Compared with MAN2A1 gene sequence of pig in NCBI database(accession No.:XM_003123823.6),the CDS region of MAN2A1 gene in Sushan pigs contained 6 single nucleotide polymorphism sites(SNPs),including 2 missense mutations and 4 synonymous mutations.Phylogenetic trees of MAN2A1 genes in different species showed that MAN2A1 gene in pigs had the closest relationship with cattle and sheep,followed by primates,and the farthest relationship with poultry and zebrafish.Phylogenetic trees of MAN2A1 genes in different pig breeds showed that,MAN2A1 gene in Sushan pigs had the closest genetic relationship with Chinese Wuzhishan pig and the farthest genetic relationship with Duroc.Bioinformatics analysis results showed that MAN2A1 protein in Sushan pigs contained 1144 amino acids,with a molecular weight of 131.42 ku and a theoretical isoelectric point of 7.85.The proportions of alpha helix,beta sheet,and random coil in secondary structure were 33.65%,15.73%and 50.61%,respectively.The GMQE score for the quality assessment of tertiary structure global model was 0.9,indicating good quality.MAN2A1 protein in Sushan pigs contained three domains(amino acids 168-498,289-503,and 649-1140).MAN2A1 protein was mainly located in the Golgi apparatus within the cytoplasm.The results of protein interaction prediction revealed that MAN2A1 protein might interact with proteins such as MAN1A1,FUT8 and COPB2.The results of tissue expression profiling showed that the expression levels of MAN2A1 gene in liver and spleen of Sushan pigs were significantly higher than those in other tissues(P<0.05),and the expression levels in heart and muscle were significantly lower than those in other tissues(P<0.05).This study successfully constructed an overexpression vector for MAN2A1 gene.Compared with control group,the expression levels of MAN2A1 gene in 3D4/21 cells were significantly upregulated in overexpression group(P<0.05).When infected with H1N1 subtype SIV,overexpression of MAN2A1 gene significantly upregulated the expression of virus M and NP genes(P<0.05).The molecular docking prediction results showed that there might be interactions between MAN2A1 protein and H1N1 subtype SIV NP,M1 and M2 proteins(iptm+ptm>0.9),indicating that MAN2A1 protein might directly interact with NP and M proteins to regulate the replication of H1N1 subtype SIV.【Conclusion】MAN2A1 gene CDS region sequence of Sushan pigs was successfully cloned in this study,and the overexpression of MAN2A1 gene promoted the replication of H1N1 subtype SIV in 3D4/21 cells.The results of this study provided a theoretical basis for further study of the function and molecular mechanism of MAN2A1 gene.
作者
戴超辉
崔乐康
李碧侠
赵为民
李辉
付言峰
李伟宁
陈彦羽
包文斌
程金花
DAI Chaohui;CUI Lekang;LI Bixia;ZHAO Weimin;LI Hui;FU Yanfeng;LI Weining;CHEN Yanyu;BAO Wenbin;CHENG Jinhua(Jiangsu Germplasm Resources Protection and Utilization Platform,Key Laboratory of Crop and Livestock Integration,Ministry of Agriculture and Rural Affairs,Institute of Animal Science,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China;ProNet Biotech(Pronetbio)Co.,Ltd.,Nanjing 210033,China)
出处
《中国畜牧兽医》
北大核心
2025年第12期6007-6019,共13页
China Animal Husbandry & Veterinary Medicine
基金
江苏省自然科学基金青年基金(BK20230753)
江苏省种业振兴揭榜挂帅项目(JBGS[2021]099)
国家生猪产业技术体系(CARS-PIG-35)
江苏省农业科学院科学仪器开放共享自主研究课题(GX(24)1004)。
