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huojinlong9287

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注册: 2008-09-28
专业: 实验动物学

[求助] 汉译英生物学方向

为了构建绿色荧光蛋白AcGFP1基因的原核表达载体pET32a-AcGFP1，并通过诱导使其在大肠杆菌中高效表达，本研究以pIRES-AcGFP1质粒为模板，采用PCR技术特异性扩增绿色荧光蛋白（AcGFP1）基因，并通过酶切和连接使其与原核表达载体pET32a（+）构成重组子，经PCR、酶切和测序鉴定后，重组质粒转化大肠杆菌Rosetta（DE3），用不同浓度的异丙基硫代半乳糖苷（IPTG）诱导表达绿色荧光蛋白，并通过15%SDS-PAGE鉴定。结果显示重组质粒pET32a-AcGFP1中的AcGFP1序列与Clontech公司的pIRES-AcGFP1质粒的AcGFP1序列完全一致，说明成功构建了含有绿色荧光蛋白AcGFP1的原核表达质粒。pET32a-AcGFP1转化大肠杆菌感受态细胞Rosetta（DE3）后，经不同浓度的IPTG诱导后均获得高效表达。SDS-PAGE验证的目的蛋白相对分子量约为44000Da，与理论预期值相符。

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power5

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专业: 环境微生物学

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爱与雨下(金币+2): 鼓励一下！~ 2011-09-09 12:08:09
huojinlong9287(金币+60, 翻译EPI+1): 翻译的不完全 2011-09-11 09:22:48
sltmac(金币+10): 2011-09-15 08:02:01
sltmac(金币+10): 2011-09-15 08:02:05
sltmac: 欢迎常来本版交流~ 2011-09-15 08:02:14

The aim of this study was to construct prokaryotic expression vector pET32a-AcGFP1, and its high level expression in E. coli cell. AcGFP1 gene was amplified from vector pIRES-AcGFP1 by PCR method, and was inserted into prokaryotic expression vector pET32a(+). The resulting pET32a-AcGFP1 was transformed into E. coli Rosetta(DE3) for expression followed by induction with isopropyl β-D-thiogalactopyranoside (IPTG). Whole-cell extracts were separated on 15% SDS-PAGE. AcGFP1 gene in pET32a-AcGFP1 was consistent with related gene in pIRES-AcGFP1 from Clontech Ltd, indicating prokaryotic expression vector with AcGFP1 gene was constructed successfully. High level expression was conducted with transformed E. coli Rosetta(DE3) after IPTG induction. A strong induction of a 44 kD protein was detected in SDS-PAGE, which was in correspondence with the theoretic value.

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