摘要
根据鸡九次跨膜超家族成员2(chicken transmembrane 9 superfamily 2,chTM9SF2)预测序列设计引物,以原代鸡胚成纤维细胞CEF、鸡成纤维细胞系DF1和鸡肝癌细胞LMH的cDNA为模板,采用PCR方法扩增chTM9SF2编码序列,并利用MEGA 7.0、Clustal W、SWISS-MODEL等生物信息学工具对鸡、鸭、人、鼠等不同物种来源的九次跨膜超家族成员2(transmembrane 9 superfamily 2,TM9SF2)进行遗传进化、同源性、结构域及结构模拟等分析;进而通过构建真核表达质粒,利用Western blot、激光共聚焦显微镜探究chTM9SF2的表达和亚细胞定位。结果显示,分别在3类细胞中均可成功获得编码序列一致的chTM9SF2基因,与禽类的同源性最高,且高度保守;该基因能够在DF-1细胞中表达,且主要定位于晚期内体或溶酶体膜。结果表明,成功扩增获得chTM9SF2基因,并在DF-1细胞成功表达,分析其定位于晚期内体或溶酶体膜,为进一步研究chTM9SF2的生理功能及在病原感染中的作用奠定了基础。
According to the reference sequence of chicken transmembrane 9 superfamily 2(chTM9SF2),the coding sequence amplification primers were designed,and cDNAs of primary chicken embryo fibroblasts(CEF),chicken fibroblast line DF1 and chicken liver cancer cell(LMH) were used as templates,and chicken TM9SF2 coding sequence was obtained from the above three templates by PCR method,and bioinformatics tools such as MEGA 7.0,Clustal W,SWISS-MODEL were used to obtain the encoding sequence of chicken,human,duck,murine TM9SF2,then the sequence was analyzed for genetic evolution,homology,domain and structural simulation.Western blot and laser confocal microscopy were used to analyze the expression and subcellular distribution of chTM9SF2.The results showed that the chTM9SF2gene with consistent coding sequence was successfully obtained in three types of cells,and the homology with bird was the highest,and the extracellular region domain was found to be highly conserved.This gene could be expressed in DF-1cells and the protein was mainly localized in the late endosomal or lysosomal membranes.The chicken-derived TM9SF2gene was successfully expressed in DF-1cells,and the protein localized in the late endosomal or lysosomal membranes,which laid a foundation for further study of the physiological function of chTM9SF2,and its role in pathogenic infection.
作者
张敏霞
池周颖
瞿孝云
刘杨
曾庆航
李昌
廖明
ZHANG Minxia;CHI Zhouyin;QU Xiaoyun;LIU Yang;ZENG Qinghang;LI Chang;LIAO Ming(National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control,College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;Ministry of Agriculture and Rural Affiars Key Laboratory for Prevention and Control for Avian Influenza and Other Major Poultry Disease,Institute of Animal Health,Guangdong Province Academy of Agricultural Sciences,Guangzhou 510640,China;Research Unit of Key Technologies for Prevention and Control of Virus Zoonoses of China Academy of Medical Science,Institute of Military Veterinary Medicine,Academy of Military Medical Science,Academy of Military Sciences,Changchun 130112,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2023年第11期2298-2307,共10页
Chinese Journal of Veterinary Science
基金
广东省科技计划资助项目(2021B1212030015)
国家自然科学基金资助项目(31972719)
国家肉鸡产业技术体系资助项目(CARS-41)
鸡肉储藏中微生物交互机制和预测模型研究资助项目(YDWS202204)
国家重点研发计划资助项目(2022YFD1801000)
“十四五”广东省农科科技创新十大主攻方向“揭榜挂帅”资助项目(2022SDZG02)。
