小鼠Fyn及其多种突变体的载体构建和蛋白生物活性预测分析

畜牧兽医学报

小鼠Fyn及其多种突变体的载体构建和蛋白生物活性预测分析

安磊，黄映雪，胡新德，张伟，闫宇华，陈树林，赵善廷^*

西北农林科技大学动物医学院，杨凌 712100

收稿日期:2012-09-03 出版日期:2013-04-23 发布日期:2013-04-23
通讯作者: 赵善廷，博导，教授，E-mail：zhaoshanting@nwsuaf.edu.cn
作者简介:安磊(1984-)，男，河南许昌人，博士研究生，主要从事分子神经生物学研究，E-mail:anlei1344@163.com
基金资助:
西北农林科技大学人才引进专项基金(Z111021101)；国家高技术研究发展计划(863计划) (2011AA10A208)

Construction of Vectors with Mouse Fyn and Its Different Mutants and Functional Prediction Analysis of Their Protein Products

AN Lei, HUANG Ying-xue, HU Xin-de, ZHANG Wei, YAN Yu-hua, CHEN Shu-lin, ZHAO Shan-ting^*

College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China

Received:2012-09-03 Online:2013-04-23 Published:2013-04-23

摘要/Abstract

摘要：

旨在构建小鼠Fyn及其多种突变体真核表达载体，预测突变后的蛋白质二级结构和功能改变。克隆小鼠大脑皮质的Fyn基因，构建克隆质粒pMD18-T-Fyn^wt，经测序验证后，以pMD18-T-Fyn^wt为模板，针对不同突变位点设计引物，构建不同突变体克隆质粒并验证，将Fyn不同的突变体克隆至真核表达载体pCAG-MCS，构建真核表达载体pCAG-MCS-Fyn^**，将表达载体转染细胞进行检测，并对其进行生物信息学分析。测序结果显示pMD18-T-Fyn^wt核苷酸序列正确率100%，突变体完全达到预期设计；pCAG-MCS-Fyn^**转染细胞后Fyn^**表达量明显升高（P<0.01）。结果表明，Fyn突变体蛋白质的二级结构与野型相比有很大改变，可能影响其生物活性。

Abstract:

Eukaryotic expression vectors of mouse Fyn and its different mutants were constructed. Structural and functional changes of their protein products were predicted. Fyn cDNA from mouse cerebral cortex was cloned and subcloned into pMD18-T. Validated pMD18-T-Fyn^wt was used as template to construct clone plasmids of different mutants using different primers. The validated Fyn^wt and its different mutants were cloned into pCAG-MCS to get the eukaryotic expression vector pCAG-MCS-Fyn^**. Characteristics of their bioinformatics were analyzed. The complete sequence showed that the cDNA sequence of Fyn^wt is identical with that in GenBank. The fragments of Fyn mutants have fully acquired as expectation. The relative expression level of multiple Fyn in cell rised obviously （P<0.01）. Bioinformatics analysis showed that the secondary structure of protein products of Fyn mutants changed remarkably by comparison with that of Fyn^wt, which may influence the bioactivity of Fyn.

中图分类号:

安磊，黄映雪，胡新德，张伟，闫宇华，陈树林，赵善廷. 小鼠Fyn及其多种突变体的载体构建和蛋白生物活性预测分析[J]. 畜牧兽医学报, doi: .

AN Lei, HUANG Ying-xue, HU Xin-de, ZHANG Wei, YAN Yu-hua, CHEN Shu-lin, ZHAO Shan-ting. Construction of Vectors with Mouse Fyn and Its Different Mutants and Functional Prediction Analysis of Their Protein Products[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

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