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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (14): 17-21.

所属专题： 生物技术

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

岩栖蝮蛇毒纤溶酶基因克隆、序列分析及真核表达载体的构建与鉴定

于德涵 赵文阁 王广慧 李贺   

  • 收稿日期:2011-02-09 修回日期:2011-03-16 出版日期:2011-06-15 发布日期:2011-06-15
  • 基金资助:

    绥化学院重点科研项目

Cloning and Sequence Analysis, Construction and Indentification of Eukaryotic Expression Vector of Fibrinolytic Enzyme from Gloydius saxatilis

  • Received:2011-02-09 Revised:2011-03-16 Online:2011-06-15 Published:2011-06-15

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摘要：

克隆岩栖蝮蛇（Gloydius saxatilis）纤维溶解酶（Fibrinolytic enzyme，FLE）基因，分析基因序列并对该基因的功能进行分析；构建真核表达载体并鉴定。从岩栖蝮蛇毒腺细胞中提取总RNA，通过RT-PCR法扩增纤溶酶基因，与pMD18-T载体连接进行TA克隆，再进行序列的测定并且对该序列进行分析。测序鉴定后将纤溶酶基因克隆入真核表达载体pEGFP-C1，构建该基因的真核表达载体pEGFP-FLE，进行双酶切、PCR和测序鉴定。扩增得到包括完整开放读码框在内的长为774 bp的纤溶酶基因序列，成功克隆了编码岩栖蝮蛇纤溶酶的基因序列，开放读码框编码257个氨基酸，其分子质量约为30 KDa，成功构建了真核表达载体pEGFP-FLE。成功克隆岩栖蝮蛇毒纤溶酶基因并构建该酶的真核表达载体为该基因的真核表达及后续研究提供依据。

关键词: 休闲农业园, 休闲农业园, 游客满意度, 结构方程模型

Abstract:

Fibrinolytic enzyme gene of Gloydius saxatilis was cloned, then analysis the gene sequence and function; construction and indentification of eukaryotic expression vector of FLE gene. Total RNA was extracted from venom cells from Gloydius saxatilis, FLE gene was amplified by RT-PCR. Then it was connected to pMD18-T vector by TA cloning. At last, sequencing and analysis the gene sequence. The FLE gene was inserted into the vector pEGFP-C1. This recombinant plasmid pEGFP-FLE was identified by digestion of endonuclease, PCR and sequencing. FLE gene was cloned successfully. Its length was 774 bp including whole open reading frame (ORF).The recombinant vector was constructed succeed. Gene sequence of FLE of Gloydius saxatilis was cloned successfully, the sequence was coding 257 animo acids and deduced its molecular weight was 30 KDa. The experiment will provide help for future research in eukaryotic expression.

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