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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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