噬菌体表面表达随机8肽文库的构建

Construction of phage random eight-peptide library

目的 :构建噬菌体表面表达随机 8肽文库 ;方法 :采用人工合成随机寡核苷酸、基因重组技术 ,噬菌体表面表达技术等 ,构建该文库 ;采用多聚酶链式反应技术、核酸杂交技术和测序分析评价鉴定。多聚酶链式反应所采用的上游引物包括载体克隆位点处部分碱基和部分外源基因的碱基 ;在核酸杂交中设计了噬菌体载体克隆位点互补探针 ,以更加准确地排除无外源基因插入的克隆 ;在测序鉴定中 ,选择了 2个混合探针杂交阳性的克隆。结果 :以上证实获得独特克隆为 2 1× 10 8;亲和富集实验证实所建噬菌体文库可以稳定扩展。结论 :我们成功地构建了噬菌体随机 8肽文库 ,其库容量为 2 1× 10 8。

AIM: To construct random eight-peptide library for the study on atherosclerosis and restenosis. METHODS and RESULTS: Random oligodeoxynucleotides encoded eight peptides were synthesized and amplified by polymerase chain reaction(PCR). The product was cloned into phage surface display vector fUSE5 in Sfi I site and electroporated into competent MC1061. The library was identified through PCR, hybridization, DNA sequencing and affinity biopanning of streptavidin. Because the upstream primer is complementary to part vector clone site sequences and part exogenous gene sequences, and the other one complementary to pIII gene of vector, thus only clones inserted exogenous gene could be amplified easily. Additionally we used the probe oligodeoxynucleotide complementary to vector clone site sequences to identify clones which were not inserted exogeneous genes. Furthermore, two hybridizing positive clones were sequenced. Their sequences are consistent with two oligodeoxynucleotide probe sequences. As a result, 2.1×108 special clones were obtained. Affinity biopanning proved that the libraries could be amplified steadily. CONCLUSION: The eight-peptide library is reliable.