中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (37): 6926-6930.doi: 10.3969/j.issn.2095-4344.2012.37.018

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

构建重组人pET-32a-瘦素高效的表达载体

谢 琳1，巫国辉2，李小林2，文辉才3   

  1. 1南昌大学第二附属医院眼科，江西省南昌市 330006
    2江西省人民医院整形颌面外科，江西省南昌市 330006
    3南昌大学第一附属医院整形美容外科，江西省南昌市 330006
  • 收稿日期:2012-04-02 修回日期:2012-07-08 出版日期:2012-09-09 发布日期:2012-09-09
  • 通讯作者: 巫国辉，副主任医师、硕士生导师，硕士，江西省人民医院整形颌面外科，江西省南昌市 330006 wu_gh@163.com
  • 作者简介:谢琳☆，女，湖南省湘潭市人，汉族，博士，主治医师。 amandaxie0510@yahoo.com.cn

Construction of a recombinant pET-32a-leptin efficient expression vector

Xie Lin1, Wu Guo-hui2, Li Xiao-lin2, Wen Hui-cai3   

  1. 1Department of Ophthalmology, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangsu Province, China
    2Department of Plastic & Dentofacial Surgery, Jiangxi Provincial People's Hospital, Nanchang 330006, Jiangxi Province, China
    3Department of Plastic & Aesthetic Surgery, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China
  • Received:2012-04-02 Revised:2012-07-08 Online:2012-09-09 Published:2012-09-09
  • Contact: Wu Guo-hui, Master, Associate chief physician, Master’s supervisor, Department of Plastic & Dentofacial Surgery, Jiangxi Provincial People's Hospital, Nanchang 330006, Jiangxi Province, China wu_gh@163.com
  • About author:Xie Lin☆, Doctor, Attending physician, Department of Ophthalmology, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangsu Province, China amandaxie0510@yahoo.com.cn

PDF

647

被引次数

3

摘要：

背景：瘦素的功能是作为脂肪细胞体脂量的信号来认识的，作为体内的能量平衡信号反馈到下丘脑，与其中各部位神经元的受体结合后参与调节进食、饮水、能量代谢。
目的：构建原核重组表达载体pET-32a-瘦素并检测其在大肠杆菌BL21中的高效表达。
方法：取人脂肪组织，RT-PCR法制备瘦素目的基因，采用DNA重组技术将其克隆至pMD18T与pET-32a原核表达载体，双酶切及测序鉴定，转化大肠杆菌BL21株诱导其表达，SDS-PAGE电泳分离鉴定，表达产物变性复性后，间接酶联免疫吸附法检测抗原反应原性。
结果与结论：实验成功扩增出520 bp的瘦素目的基因并构建了原核重组表达载体pET-32a-瘦素；测序结果与GenBank收录序列相一致；目的蛋白pET-32a-瘦素呈极高效表达，占总蛋白的50%以上；变性复性后抗原反应原性较复性前明显提高(P < 0.01)。结果证实，实验成功构建原核重组高效表达的载体pET-32a-瘦素，并提高其抗原反应原性。

关键词: 瘦素, 重组, 变性, 复性, 克隆, 原核表达, 抗原反应原性, RT-PCR\酶联免疫吸附, 组织工程

Abstract:

BACKGROUND: Leptin is recognized as a feedback signal of body fat by bonding receptor in the hypothalamus, which is related to ingestion, drinking, and energy metabolism.
OBJECTIVE: To construct a prokaryotic recombinant expression plasmid pET-32a-leptin and to analyze its expression in BL21 Escherichia coli.
METHODS: The adipose tissues were obtained, and leptin gene was obtained by reverse transcription-PCR. The target gene was orientating cloned into pMD18T and pET-32a vector by DNA recombinant technical. Position clones were transformed into BL21 Escherichia coli and expressed by double digestion and DNA sequencing. Then the expression product was detected for the antigen reactionogenicity after degeneration and renaturation.
RESULTS AND CONCLUSION: 520 bp leptin fragment was amplified and the prokaryotic recombinant expression plasmid pET-32a-leptin was correctly constructed. The Leptin gene fragment in position clones was tested be same as the sequence of the GenBank public by DNA sequencing. The position protein pET-32a-leptin was highly expressed and about 50% on total protein. The antigen reactionogenicity of expression product was enhanced after degeneration and renaturation (P < 0.01). Prokaryotic recombinant expression plasmid pET-32a-leptin was successfully constructed and the target protein was expressed largely.

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