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huojinlong9287银虫 (小有名气)
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英文精细润色 生物学 （已附中文）
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润色英文翻译 请不要灌水 高手优先 为了构建猪SRY基因的原核表达载体pET-32a(+)-SRY，并通过诱导使其在大肠杆菌中获得高效表达。本研究采用添加限制性内切酶位点的引物特异性扩增SRY基因，连入pMD19-T simple载体，转化入大肠杆菌DH5α，克隆后提取pMD19-T-SRY阳性重组质粒，使用相同的内切酶同时对pMD19-T-SRY质粒和原核表达pET-32a（+）载体进行酶切，连接后使SRY基因定向克隆到pET-32a(+)表达载体中。经PCR、酶切和测序鉴定后，重组质粒转化大肠杆菌感受态DH5α，提取质粒后再次转化大肠杆菌Rosetta（DE3），用不同浓度的异丙基硫代半乳糖苷（IPTG）诱导表达，并通过15% SDS-PAGE鉴定。结果显示，不同浓度IPTG诱导的SRY基因均在大肠杆菌中进行了高效特异性融合表达。 To construct prokaryotic expression vector pET-32a(+)-SRY of pig and induced it expresses efficiently in E. coli cell, SRY gene was amplified by using primer with restriction enzymes, and it was inserted into pMD19-T simple vector and transferred into the bacterium DH5α for replication. The pMD19-T-SRY recombinant plasmids were extracted, then pMD19-T-SRY and pET-32a（+）were digested with same restriction enzymes. By this directed cloning technique, the SRY gene was inserted into pET-32a（+）expression plasmid. After identified by PCR, restriction enzyme digestion and sequencing, The pMD19-T-SRY recombinant plasmids were transformed into competent cell DH5α. the pMD19-T-SRY plasmids were extracted and transformed to competent cell Rosetta（DE3）. pMD19-T-SRY was induced with different concentrations of isopropyl-β-D-thiogalactopyranoside (IPTG) and detected on 15% SDS-PAGE. The results showed that the fusion protein were specifically high efficient expression in SDS-PAGE after induced by different concentrations of IPTG. |
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xiaokaizi
金虫 (著名写手)
- 翻译EPI: 39
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爱与雨下(金币+2): ！~ 2011-10-09 20:29:47
huojinlong9287(金币+100, 翻译EPI+1): 2011-10-10 09:59:00
爱与雨下(金币+2): ！~ 2011-10-09 20:29:47
huojinlong9287(金币+100, 翻译EPI+1): 2011-10-10 09:59:00
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谈不上高手，只是相互交流学习一下。注意有几处修改的地方（上传时标记被抹去了）。仅供参考，， To construct pig’s prokaryotic expression vector pET-32a(+)-SRY and induce it to express efficiently in E. coli cell, SRY gene was amplified by (using) primer with restriction enzymes, and it was inserted into pMD19-T simple vector and transferred into the bacterium DH5α .After replication the pMD19-T-SRY recombinant plasmids were extracted, then pMD19-T-SRY and pET-32a（+）were digested by the same restriction enzymes at the same time. By this directed cloning technique, the SRY gene was inserted into pET-32a（+）expression plasmid. After PCR identifying, restriction enzyme digestion and sequencing testing, the pMD19-T-SRY recombinant plasmids were transformed into competent cell DH5α. The pMD19-T-SRY plasmids were extracted and transformed to competent cell Rosetta(DE3). pMD19-T-SRY was induced with different concentrations of isopropyl-β-D-thiogalactopyranoside (IPTG) and identified on 15% SDS-PAGE. The results showed that the fusion protein had high efficient expression in SDS-PAGE when induced by different concentrations of IPTG. |
2楼2011-10-09 20:19:54
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