牛METTL3真核表达载体的构建及生物信息学分析  

Construction and bioinformatics analysis of eukaryotic expression vector of bovine METTL3

作　　者：黄雯静 赵尚琪 吴陈华 高丽 付金蕾 刘思雨 赵童 陈楠楠[1] 岳山[1] 张泽财[1] 刘宇[1] 周玉龙[1] 朱战波[1] HUANG Wen-jing;ZHAO Shang-qi;WU Chen-hua;GAO Li;FU Jin-lei;LIU Si-yu;ZHAO Tong;CHEN Nan-nan;YUE Shan;ZHANG Ze-cai;LIU Yu;ZHOU Yu-long;ZHU Zhan-bo(School of Animal Science and Technology,Heilongjiang Bayi Land Reclamation University,Engineering Research Center for Cattle Disease Prevention and Control of Heilongjiang Province,Daqing 163319,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江省牛病防控工程技术研究中心,黑龙江大庆163319

出　　处：《生物技术》2021年第6期527-533,共7页Biotechnology

基　　金：国家自然科学基金项目(“NCP型BVDV急性感染中BTLA调控CD8+TLs活化、增殖及杀伤功能的机制研究”,32072896);黑龙江省牛病防制重点实验室开放课题(PCBD201705);黑龙江八一农垦大学研究生创新科研项目(YJSCX2018-Y26,YJSCX2019-Y31)。

摘　　要：[目的]对牛甲基转移酶METTL3基因进行真核表达并对其蛋白进行生物信息学分析。[方法]以MDBK细胞为模板,RT-PCR扩增牛METTL3基因片段,构建pcDNA3.0-METTL3载体并转染至293T细胞后,进行Western Blotting验证,通过生物信息学在线工具分析METTL3蛋白。[结果]PCR、双酶切鉴定、Western Blotting和生物信息学分析表明牛METTL3基因编码区序列全长为1760 bp,编码了580个氨基酸,蛋白的分子质量64.4469 kDa,PI为5.98,属于不稳定亲水性蛋白,不存在跨膜结构与信号肽。二级结构主要是由α螺旋和无规则卷曲构成,三级结构与二级结构预测结果一致。[结论]成功构建了pcDNA3.0-HA-METTL3真核表达载体,生物信息学分析表明,牛METTL3蛋白为在细胞质中分泌合成的可溶蛋白,α螺旋和无规则卷曲为二级结构中最主要的结构元件。[Objective]Eukaryotic expression of bovine METTL3 gene and bioinformatics analysis of its protein.[Method]Using MDBK cells as a template,RT-PCR amplifies bovine METTL3 gene fragments,constructs pcDNA3.0-METTL3 vector and transfects it into 293 T cells,then performs Western Blotting verification,and analyzes METTL3 protein with bioinformatics online tools.[Result]PCR,double restriction enzyme digestion,Western Blotting and bioinformatics analysis showed that the full length of the coding region of the cattle METTL3 gene was 1760 bp,encoding 580 amino acids,and the molecular mass of the protein was 64.4469 kDa,and the PI was 5.98.Stable hydrophilic protein,without transmembrane structure and signal peptide.The secondary structure is mainly composed ofαhelices and random coils,and the tertiary structure is consistent with the prediction results of the secondary structure.[Conclusion]The eukaryotic expression vector pcDNA3.0-HA-METTL3 was successfully constructed.Bioinformatics analysis shows that the bovine METTL3 protein is a soluble protein secreted and synthesized in the cytoplasm,andαhelix and random coil are the most important structural elements in the secondary structure.

关 键 词：牛METTL3基因 真核表达载体 生物信息学 分析和预测 

分 类 号：S855.3[农业科学—临床兽医学]