以Trx为骨架构建可诱导表达的酵母构象型随机肽库

Construction of an inducible yeast random conformation-constrained peptide library based on Trx scaffold

目的:以硫氧还蛋白(Trx)为骨架,pB ridge为载体,构建可诱导表达的酵母构象型随机10肽库。方法:利用PCR扩增出trx基因,向trx的突环区引入AscⅠ酶切位点,构建pB ridge-mTrx重组载体。采用(NNK)10的编码方式设计并合成两条寡核苷酸链,经退火、延伸,AscⅠ,RsrⅡ双酶切后,克隆至pB ridge-mTrx载体,电转化E.coliDH10B感受态细胞。对所构建多肽文库的库容和随机性进行了鉴定,并利用W estern印迹对文库的可诱导性进行分析。结果:获得库容量为8.7×108的随机肽库。随机挑选20个克隆进行测序,4个核苷酸的出现频率与理论值接近。在M et缺陷培养基中,随机肽库可被诱导表达。结论:成功构建可诱导表达的随机肽库,为进一步筛选特异性多肽奠定了基础。

Objective： To construct an inducible yeast decamer random conformation-constrained peptide library based on thioredoxin （Trx） scaffold in pBridge vector. Methods： Trx gene was obtained by PCR amplification. Asc I site was inserted into the active site loop of trx. Gene pool encoding peptide library with decamer random amino acids was generated by two complementary oligonucleotides. After annealing, elongation and Asc Ⅰ and Rsr Ⅱ digestion, the DNA fragment was inserted into pBridge-mTrx vector. Ligation mixtures were electroporated into the competent E. coli DH10B cells. The titer and randomness of the library was identified and the inducible expression of the library was analyzed by Western blot. Results ： A random peptide library with approximately 8.7 × 10^8 independent transformants was obtained. Twenty clones from the peptide library were chosen randomly and their sequences were determined, As a result, the observed frequencies of nucleotides were not significantly different from the expected frequencies. The peptide library was conditionally expressed in the absence of methionine in the medium. Conclusion： The inducible peptide library we conducted achieves destination, which lays foundation for the further screening of the specific peptide.