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-Fynwt,经测序验证后,以pMD18-T-Fynwt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-Fynwt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。
Eukaryotic expression vectors of mouse Fyn and its different mutants were construc- ted. 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-Fynwt was used as template to construct clone plasmids of different mutants using different primers. The validated Fynwt and its different mutants were cloned into pCAG-MCS to get the eukaryotic ex- pression vector pCAG-MCS-Fyn＊＊. Characteristics of their bioinformatics were analyzed. The complete sequence showed that the cDNA sequence of FynWt 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 seconda- ry structure of protein products of Fyn mutants changed remarkably by comparison with that of Fynwt, which may influence the bioactivity of Fyn.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2013年第4期635-641,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
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)
