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fbook

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利用大肠杆菌的偏爱密码子，把三个LNK-16串联起来，根据串联起来的氨基酸残基，通过反向翻译出它的目的基因片段。在目的基因片段两端加入限制性酶切位点和保护碱基，利用SOEingPCR的方法把目的基因片段分成四段互补的引物。人工这合成这四段引物，通过TD-PCR方法最终扩增出目的基因。把目的基因和质粒载体PET30a(+)进行双酶切后，利用T4DNA连接酶重新构建出质粒载体PET30a(+)-LNK-16，通过双酶切和PCR鉴定成功构建了载体，经测序表明与设计的目的基因完全相同。
把含有测序完全正确载体的菌体大量表达，经ITPG诱导后，在大肠杆菌包涵体中表达出目的蛋白，目的蛋白纯化后在三乙醇胺缓冲液中用激肽释放酶酶切，酶解液对大肠杆菌表现出抗菌活性。从而为抗菌肽不能在原核中表达提供的解决的方法，为解决抗菌肽来源问题的研究奠定了一定的基础。

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1楼 2011-04-07 08:11:04

已阅回复此楼关注TA 给TA发消息送TA红花 TA的回帖

zs626

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fbook(金币+150, 翻译EPI+1): 十分感谢你提的宝贵意见…… 2011-04-09 08:14:25

We connected three LNK-16 peptides by using codon preference of E.coli, and took a reverse translation of its target gene according to the amino acid residues linked together. By adding restriction enzyme sites and protective bases at both ends of the target gene, we divided the target gene into four complementary primers through the method of SOEingPCR. Then we synthesized these four primers and amplified the target gene by TD-PCR. After double digestion of the target gene and plasmid vector PET30a (+), we re-constructed the plasmid vector PET30a (+)-LNK-16 with T4DNA ligase. With double digestion and PCR identification we successfully constructed the vector, and sequencing showed that it was the same as the target gene we designed.

After taking mass expression of vector proved to be completely correct by sequencing and ITPG induction, we expressed the target protein in cytorrhyctes of E.coli. After purification, the protein was digested by kallikrein in triethanolamine buffer, and the enzymolysis liquid showed antibacterial activity to E.coli. Thus, the research provided new method for solving the problem of antimicrobial peptides can not be expressed in prokaryote and laid a foundation for solving the source problem of antibacterial peptides.

备注：
1、原文“把含有测序完全正确载体的菌体大量表达”表达似有错误，“表达菌体”描述有逻辑问题；
2、原文“从而为抗菌肽不能在原核中表达提供的解决的方法”似应改为“从而为抗菌肽不能在原核中表达提供了解决的方法”。
3、水平有限，仅供参考，谢谢。

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