上海交通大学学报（医学版）

›› 2013, Vol. 33 ›› Issue (4): 425-.doi: 10.3969/j.issn.1674-8115.2013.04.009

• 论著（基础研究） • 上一篇    下一篇

血红素氧合酶-1基因真核表达载体的构建及其在大鼠腹膜间皮细胞中的表达

朱 淳1, 郭志勇2, 刘怡晟1, 黄海东1, 蒋更如1   

  1. 1.上海交通大学 医学院附属新华医院肾内科, 上海 200092； 2.第二军医大学附属长海医院肾内科, 上海 200433
  • 出版日期:2013-04-28 发布日期:2013-05-03
  • 通讯作者: 郭志勇, 电子信箱: chguozhiyong@126.com。
  • 作者简介:朱 淳(1964—）, 女, 主任医师, 博士生； 电子信箱: zhuchun26@sohu.com。
  • 基金资助:

    上海市卫生局基金（20114335)

Construction of eukaryotic expression vector of heme oxygenase-1 and its expression in rat peritoneal mesothelial cells

ZHU Chun1, GUO Zhi-yong2, LIU Yi-sheng1, HUANG Hai-dong1, JIANG Geng-ru1   

  1. 1.Department of Nephrology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China； 2.Department of Nephrology, Changhai Hospital Affiliated to the Second Military Medical University of PLA, Shanghai 200433, China
  • Online:2013-04-28 Published:2013-05-03
  • Supported by:

    Shanghai Municipal Health Bureau Foundation, 20114335

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摘要：

目的 构建大鼠血红素氧合酶-1(HO-1)真核表达载体，并以该重组表达载体体外转染培养的大鼠腹膜间皮细胞(RPMCs）。方法 从SD大鼠脾脏中提取总RNA，并经RT-PCR扩增HO-1基因的CDS区全长片段，将PCR产物克隆至pMD-18T载体，BamHⅠ和KpnⅠ双酶切后获取HO-1片段，用T4 DNA连接酶将HO-1片段与双酶切后的真核表达载体pcDNA3-1 (－)连接，构建大鼠HO-1真核表达载体pcDNA3-1 (－)-HO-1。酶切鉴定和测序验证后，利用脂质体介导将重组质粒转染原代培养的RPMCs，通过RT-PCR和Western blotting检测RPMCs中HO-1的表达。结果 成功克隆了大鼠HO-1基因并构建了其真核表达载体pcDNA3-1 (－)-HO-1，酶切鉴定证实基因正确地插入表达载体中，并经DNA测序验证。pcDNA3-1 (－)-HO-1转染RPMCs后，经RT-PCR和Western blotting检测，HO-1在RPMCs中获得高效表达。结论 HO-1基因真核表达载体的成功构建以及HO-1在RPMCs中的高效表达，为进一步研究HO-1在腹膜透析所致RPMCs损伤中的作用奠定了基础。

关键词: 血红素氧合酶-1, 表达载体, 腹膜间皮细胞, 大鼠

Abstract:

Objective To construct the eukaryotic expression vector of heme oxygenase-1 (HO-l) gene, and transfect the recombinant expression vector to rat peritoneal mesothelial cells (RPMCs) in vitro. Methods Total RNA was extracted from SD rat spleen, and the full length fragment of HO-1 CDS part was amplified by RT-PCR. The product of PCR was cloned to pMD-18T vector. The pMD-18T-HO-1 and pcDNA3-1 (－) vector were digested with restriction endonuclease BamHⅠ and KpnⅠ, then T4 DNA ligase was used to ligate HO-1 gene fragment and digested pcDNA3-1 (－) vector to construct the eukaryotic expression vector pcDNA3-1 (－)-HO-1. The recombinant plasmid pcDNA3-1 (－)-HO-1 was transfected into RPMCs. The expression of HO-1 in RPMCs was detected by RT-PCR and Western blotting respectively. Results The full length of rat HO-1 gene was cloned, and the eukaryotic expression vector pcDNA3-1 (－)-HO-1 was successfully constructed. Restriction endonucleases digestion confirmed the target DNA fragment was inserted into the expression vector, and was verified by DNA sequencing. RT-PCR and Western blotting revealed that HO-1 was highly expressed in RPMCs after transfection with pcDNA3-HO-1. Conclusion HO-1 eukaryotic expression vector has been successfully constructed, and HO-1 expresses effectively in RPMCs, which lays a foundation for the further study of the effect of HO-1 in RPMCs injury during peritoneal dialysis.

Key words: heme oxygenase-1, expression vector, peritoneal mesothelial cell, rat