【作者】 黄海明；

【导师】 高友鹤；

【作者基本信息】 中国协和医科大学 ， 病理生理学， 2004， 博士

【摘要】 本部分介绍了一种新的随机多肽文库的构建方法，即用基因组DNA为原料来构建随机多肽文库。 一般认为，基因组DNA是不能够直接用来表达出有生物学意义的蛋白质或多肽。但是，如果我们需要的是随机多肽，基因组DNA就可以被认为是非常有用的资源。当大的基因组DNA被仅识别4个核苷酸的限制性内切酶酶切后，产生的短片段DNA可以近似的认为是随机序列的片段，这样的短的随机片段在本实验中被用来直接表达随机多肽，成为随机多肽文库。例如人的基因组DNA为2.91×10~9bp，这样大的基因组DNA经识别4个核苷酸的限制性内切酶(DpnⅡ或Tsp509I)酶切后，因为这种酶在基因组上平均每隔256bp即有一个酶切位点，因此可以产生约10~7(2.91×10~9／256)个片段。由于每个片段的平均长度为256bp，所以这样的片段可以编码85个氨基酸(256／3)，又由于每64个密码子当中有3个终止密码子，所以最后这样的片段表达出的多肽的平均长度为21个氨基酸(64／3)。这些片段可以克隆到任何表达载体中。在这里我们将这些片段连接到用DpnⅡ(Tsp509I)的同尾酶BamHI(EcoRI)预先酶切的酵母双杂交载体pGADT7上后，电击转化大肠杆菌形成随机多肽文库。 在论文中，我们构建了两个这样的随机多肽文库。一个是用DpnⅡ酶切人基因组DNA，所构建的文库的容量为1.1×10~6cfu。另一个是用Tsp509I酶切人基因组DNA，所构建的文库的容量为1.4×10~7cfu。更多还原

【Abstract】 A novel method for making random peptide library with genomic DNA was introduced in the first part of this dissertation. It is generally believed that genomic DNA cannot be directly used for expression of biological meaningful proteins or peptides. However, if we are to make random peptide library, genomic DNA can be considered a useful DNA fragment sources. Large genomic DNAs can be digested to short fragments and those short fragments of DNA can be considered nearly random and were used here for making random peptide library. For example, the human genomic DNA is 2.91× 10~9bp and can produce 10~7(2.91 × 10~9/256) fragments after it is fully digested with restriction endonuclease DpnII or Tsp509I, which recognizes 4 nucleotides. These fragments, which are 256bp long in average and can be cloned into any expression vectors, for example yeast two-hybrid vector pGADT7, predigested by BamHI or EcoRI, which generates compatible cohesive ends with DpnII or Tsp509I respectively. In those libraries, average fragment codes 85(256/3) amino acids. Since there are 3 stop codons in every 64 codons, the peptides expression stop at average length of 21 amino acid residues.In the first part of the dissertation, two such random peptide libraries were constructed, one was human genomic DNA digested by DpnII, in which 1.1 × 10~6 independent clones were obtained while the other was digested by Tsp509I, in which 1.4 ×10~7 independent clones were obtained.更多还原