《植物生理学报》 2010, 46(2): 108-112

转基因香石竹中 F3 ’ 5 ’H 基因的克隆、表达和免疫学鉴定

白蓝^1,2, 贾军伟^1,*, 孙建萍¹, 李鹏¹, 赵明文², 潘爱虎^1,**

1 上海市农业科学院生物技术研究所, 上海市农业遗传育种重点实验室, 农业部转基因植物环境安全监督检验测试中心(上 海), 上海 201106; 2 南京农业大学生命科学学院, 农业部农业环境微生物工程重点开放实验室, 南京 210095

通信作者：贾军伟；E-mail: aihup@yahoo.com.cn；Tel: 021-62208750

摘　要：

在研究转基因香石竹品系月之霓裳(Moonshade)、月之伊人(Moonlite)中外源基因F3’5’H的表达中, 本文克隆了F3’5’H 全长基因 1.5 kb, 构建获得工程菌株Escherichia coli BL21(DE3) (+F3'5'H)。SDS-PAGE 分析的结果显示, 该菌株高效表达出 F3’5’H重组蛋白, 约占菌体总蛋白的30%。用经纯化的F3’5’H重组蛋白作为抗原, 制备F3’5’H重组蛋白的抗血清, 经ELISA 免疫学分析表明, 该抗血清的效价为 1:25 600。Western blot 结果表明 F3’5’H 重组蛋白具有良好的 IgG 结合活性, 且抗血清 与转基因香石竹品系月之霓裳和月之伊人中的外源基因F3’5’H 所表达的蛋白发生明显的抗原抗体反应。这样, 月之霓裳 和月之伊人用于评价转基因香石竹品系的环境安全性在我国也得到了验证。

关键词：转基因香石竹; F3’5’H; 原核表达; 免疫学鉴定

收稿：2009-07-30   修定：2009-12-30

资助：上海市科技兴农重点攻关项目(沪农科攻字2008 第8-9 号)

Cloning, Expression and Immunological Identification of F3’5’H Gene in Transgenic Carnation (Dianthus canpphyllus Linn.)

BAI Lan^1,2, JIA Jun-Wei^1,*, SUN Jian-Ping¹, LI Peng¹, ZHAO Ming-Wen², PAN Ai-Hu^1,**

¹Biotech Research Institute of Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Supervision, Inspection and Test Center for Environment Safety of GM Crops of MOA (Shanghai), Shanghai 201106, China; ²College of Life Sciences, Nanjing Agricultural University, Key Lab of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Nanjing 210095, China

Corresponding author: JIA Jun-Wei; E-mail: aihup@yahoo.com.cn; Tel: 021-62208750

Abstract:

In order to study the expression of exogenous gene F3’5’H in transgenic carnation lines Moonshade and Moonlite, the full-length F3’5’H gene of 1.5 kb was cloned, and the strain Escherichia coli BL21(DE3)(+F3’5’H) was obtained. SDS-PAGE analysis showed that the high-level expressed recombinant protein F3’5’H was detected and the expression level was almost 30% of the total protein. The recombinant protein was used as antigen to obtain the antiserum. And the immunological analysis of ELISA showed the antiserum titer was 1:25 600. Western blot analysis indicated that the recombinant protein F3’5’H could combine with sera IgG, and the antiserum showed specific immunological response to the F3’5’H protein extracted from Moonshade and Moonlite.

Key words: transgenic carnation (Dianthus canpphyllus); F3’5’H; prokaryotic expression; immunological identification