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huojinlong9287銀蟲 (小有名氣)
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[求助]
請(qǐng)高手潤(rùn)色英文翻譯 生物學(xué)
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為了構(gòu)建綠色熒光蛋白AcGFP1基因的原核表達(dá)載體pET32a-AcGFP1，并通過(guò)誘導(dǎo)使其在大腸桿菌中高效表達(dá)，本研究以pIRES-AcGFP1質(zhì)粒為模板，采用PCR技術(shù)特異性擴(kuò)增綠色熒光蛋白（AcGFP1）基因，并通過(guò)酶切和連接使其與原核表達(dá)載體pET32a（+）構(gòu)成重組子，經(jīng)PCR、酶切和測(cè)序鑒定后，重組質(zhì)粒轉(zhuǎn)化大腸桿菌Rosetta（DE3），用不同濃度的異丙基硫代半乳糖苷（IPTG）誘導(dǎo)表達(dá)綠色熒光蛋白，并通過(guò)15% SDS-PAGE鑒定。結(jié)果顯示重組質(zhì)粒pET32a-AcGFP1中的AcGFP1序列與Clontech公司的pIRES-AcGFP1質(zhì)粒的AcGFP1序列完全一致，說(shuō)明成功構(gòu)建了含有綠色熒光蛋白AcGFP1的原核表達(dá)質(zhì)粒。pET32a-AcGFP1轉(zhuǎn)化Rosetta（DE3），經(jīng)不同濃度的IPTG誘導(dǎo)，SDS-PAGE檢測(cè)均獲得高效表達(dá)。 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】應(yīng)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樓2011-10-02 16:12:17
kakarote
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2樓2011-10-02 15:58:05
xiaokaizi
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【答案】應(yīng)助回帖
huojinlong9287(金幣+50, 翻譯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樓2011-10-02 16:12:34
xiaokaizi
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5樓2011-10-02 16:14:52
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