【摘要】 目的 基于定向进化的新特性蛋白质合成研究的关键在于基因变体库的生成以及目标蛋白质的筛选，而目标蛋白质筛选的成功率取决于基因变体库的多样性，针对采用从头寡核苷酸合成策略的基于基因芯片技术的基因变体库合成生物技术，本文研究并设计一种旨在提高基因变体库多样性的寡核苷酸设计方法。方法 采用了逆翻译算法提高基因序列的一致性，并利用在高同源区域的片段切割来提高基因合成过程中的重组率，从而提高基因变体库的多样性。结果 与DNAWorks和TmPrime设计后的基因序列相比，本文方法能够达到更高的一致性，同时基因片段切割结果显示该方法能够使切割后的前后片段末端具有非常高的相似度。最后设计的寡核苷酸经过基因芯片合成筛选得到了目标蛋白质。结论 本文提出的寡核苷酸设计方法充分利用了DNA序列间的基因重组，设计出的寡核苷酸序列经过基因芯片的合成证实了可行性，说明本文提出的寡核苷酸设计算法能够有效提高基因变体库的多样性。更多还原

【Abstract】 Objective The key to the research of synthesis of protein with new characteristics based on directed evolution lies in the generation of library of variants and the screening of target proteins. The success rate of target protein screening depends on the diversity of library of variants. Therefore, for the synthetic biotechnology of library of variants based on de novo oligonucleotide synthesis strategy and gene chip technology, we design and develop an oligonucleotide design tool to improve the diversity of library of variants. Methods We first apply the reverse translation algorithm to improve the identity of the gene sequences, and use the fragment cutting in the highly homologous region to improve the recombination rate in the process of gene synthesis, in order to increase the diversity of library of variants. Results Compared with the gene sequences designed by DNAWorks and TmPrime, our method can achieve higher identity. At the same time, the long gene sequences cutting results show that we can make the ends of the fragments after the cutting process have a high similarity. Finally, the designed oligonucleotides are synthesized and screened by gene chip to obtain the target protein. Conclusions The oligonucleotide design method proposed in this paper makes full use of the genetic recombination between DNA sequences. The designed oligonucleotide sequences synthesized by gene chip verifies the feasibility of our method and illustrates the effectiveness of increasing the diversity of library of variants.更多还原