四川农业大学学报 ›› 2019, Vol. 37 ›› Issue (02): 215-220.doi: 10.16036/j.issn.1000-2650.2019.02.010

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版纳微型猪近交系B4GALNT2基因真核表达载体构建及亚细胞定位分析

王淑燕1, 宋雪1, 王配1, 霍海龙2, 张霞1, 张永云3, 李罗刚1, 王雪飞1, 霍金龙1,*   

  1. 1.云南农业大学动物科学技术学院/云南省版纳微型猪近交系重点实验室,昆明 650201;
    2.云南农业职业技术学院教务处,昆明 650031;
    3.云南农业大学农科专业基础实验教学示范中心,昆明 650201
  • 收稿日期:2018-12-03 出版日期:2019-04-28 发布日期:2021-01-29
  • 作者简介:王淑燕,硕士,实验师,主要从事异种器官移植免疫排斥基础研究工作。*责任作者:霍金龙,博士,副教授,主要从事异种器官移植免疫排斥基础研究工作,E-mail: jinlonghuo973@163.com。
  • 基金资助:
    国家自然科学基金项目(31660650); 云南省应用基础研究计划面上项目(2017FB066)

Construction of Eukaryotic Expression Vector and Subcellular Localization Analysis of B4GALNT2 Gene in Banna Mini-Pig Inbred Line

WANG Shuyan1, SONG Xue1, WANG Pei1, HUO Hailong2, ZHANG Xia1, ZHANG Yongyun3, LI Luogang1, WANG Xuefei1, HUO Jinlong1,*   

  1. 1. Faculty of Animal Science and Technology/Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province,Yunnan Agricultural University,Kunming 650201,China;
    2. Teaching Affairs Department,Yunnan Vocational and Technical college of Agriculture,Kunming 650031,China;
    3. Teaching Demonstration Center of the Basic Experiments of Agricultural Majors,Yunnan Agricultural University,Kunming 650201,China
  • Received:2018-12-03 Online:2019-04-28 Published:2021-01-29

摘要: 【目的】研究异种器官移植免疫排斥相关基因B4GALNT2的亚细胞定位情况。【方法】以版纳微型猪近交系(BMI)为试验对象,通过RT-PCR方法获得B4GALNT2基因cDNA序列,运用分子克隆技术构建B4GALNT2和报告基因EGFP的重组真核表达载体pEGFP-C1-B4GALNT2,将其转染猪肾上皮细胞PK15进行瞬时表达,同时用Mito Tracker和Hoechst33342荧光染料分别对线粒体和细胞核染色,通过EGFP示踪检测B4GALNT2在PK15细胞中的表达和定位。【结果】成功构建了BMI B4GALNT2基因的绿色荧光蛋白融合表达载体pEGFP-C1-B4GALNT2,转染PK15细胞后主要在细胞质中检测到绿色荧光蛋白的表达,试验结果与PSORTⅡserver网站的预测一致。【结论】B4GALNT2蛋白主要定位于细胞质,揭示该蛋白主要在细胞质中发挥其功能作用。

关键词: 版纳微型猪近交系, β1, 4-N-乙酰氨基半乳糖转移酶 2(B4GALNT2), Sda抗原, 真核表达, 亚细胞定位

Abstract: 【Objective】 The purpose of this study was to determine the subcellular localization of B4GALNT2 gene related immune rejection in xenotransplantation. 【Method】 The cDNA sequence of B4GALNT2 gene was obtained by RT-PCR using Banna minipig inbred line (BMI) as experimental animal. The recombinant eukaryotic expression vector pEGFP-C1-B4GALNT2 of the B4GALNT2 and reporter gene EGFP were constructed by molecular cloning technique. pEGFP-C1-B4GALNT2 was transfected into porcine kidney cells (PK15) for transient expression, then the nucleus and mitochondria were stained with Mito Tracker and Hoechst 33342 fluorescent dyes respectively. Finally, the expression and localization of B4GALNT2 in PK15 cells were traced by using EGFP. 【Result】 The green fusion expression vector pEGFP-C1-B4GALNT2 of fluorescent protein of BMI B4GALNT2 gene was successfully constructed and the expression of green fluorescent protein was detected mainly in the cytoplasm after transfecting PK15 cells, which was consistent with the prediction of PSORT II server website. 【Conclusion】 B4GALNT2 protein is mainly located in the cytoplasm, revealing it plays a major role in the cytoplasm.

Key words: Banna Mini-Pig Inbred Line, β1, 4-N-acetylgalactosyltransferase2(B4GALNT2), Sda antigen, eukaryotic expression, subcellular localization

中图分类号: 

  • S828.2