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huojinlong9287银虫 (小有名气)
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[求助]
请高手润色英文翻译 生物学
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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Rosetta（DE3），经不同浓度的IPTG诱导，SDS-PAGE检测均获得高效表达。 The aim of this study was to construct prokaryotic expression vector pET32a-AcGFP1 and induced it high efficient expression in E. coli cell. AcGFP1 gene of green fluorescent protein was amplified from pIRES-AcGFP1 plasmid by PCR method, and was inserted into prokaryotic expression vector pET32a(+) by restriction enzyme digestion. After identifying by PCR, restriction enzyme digestion and sequencing method, AcGFP1 gene was induced with different concentrations of isopropyl-β-D-thiogalactopyranoside (IPTG) and detected on 15% SDS-PAGE. The results showed that the sequence of AcGFP1 gene in pET32a-AcGFP1 recombinant plasmid was consistent with AcGFP1 gene in pIRES-AcGFP1 plasmid from Clontech Ltd. The prokaryotic expression vector with AcGFP1 gene of green fluorescent protein was constructed successfully. All the pET32a-AcGFP1 plasmids transformed in E. coli Rosetta(DE3) were high efficient expression in SDS-PAGD after inducing with different concentrations IPTG. |
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xiaokaizi
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【答案】应助回帖
| To construct prokaryotic expression vector pET32a-AcGFP1 and make it expresses efficiently in E. coli cell, AcGFP1 gene of green fluorescent protein was amplified from pIRES-AcGFP1 plasmid with PCR method, and it was inserted into prokaryotic expression vector pET32a(+) by restriction enzyme digestion. After identified by PCR, restriction enzyme digestion and sequencing, AcGFP1 gene was induced with different concentrations of isopropyl-β-D-thiogalactopyranoside (IPTG) and checkered on 15% SDS-PAGE. |
3楼2011-10-02 16:12:17
kakarote
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2楼2011-10-02 15:58:05
xiaokaizi
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huojinlong9287(金币+50, 翻译EPI+1): 2011-10-02 16:25:45
| The results showed that the sequence of AcGFP1 gene in pET32a-AcGFP1 recombinant plasmid was consistent with AcGFP1 gene in pIRES-AcGFP1 plasmid from Clontech Ltd, proving that the prokaryotic expression vector with AcGFP1 gene of green fluorescent protein was constructed successfully. And all of the pET32a-AcGFP1 plasmids transformed in E. coli Rosetta(DE3) were high efficient expression in SDS-PAGD after induced by different concentrations of IPTG. |
4楼2011-10-02 16:12:34
xiaokaizi
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5楼2011-10-02 16:14:52
