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目的获得表面展示有前列腺酸性磷酸酶多肽片段(PAP_(114-128))的PP7噬菌体样颗粒,并证实其保留原有的生物活性。方法用基因突变和重叠PCR技术扩增出PP7衣壳蛋白单链二聚体(简称为2PP7)基因,将其插入到载体p ETDuet-1中,获得质粒p ETDuet-2PP7,然后通过普通PCR获得携带有PAP_(114-128)编码基因的PP7衣壳蛋白基因,将其插入到质粒p ETDuet-2PP7中,获得质粒p ETDuet-2PP7-PAP_(114-128);将上述重组质粒转化大肠杆菌,诱导表达产物用SDS-PAGE、双向免疫扩散以及ELISA进行鉴定。结果以ExTaq DNA聚合酶行重叠PCR可获得2PP7基因,其表达产物衣壳蛋白单链二聚体与野生型PP7噬菌体衣壳蛋白的抗原性相同;展示于PP7噬菌体样颗粒表面的PAP_(114-128)表位与天然的人PAP蛋白相应表位无异,且能诱导较高水平的抗体产生。结论含重复序列的2PP7基因可通过将基因突变和重叠PCR技术联用获得,且以其为基础成功制备表面展示有PAP114-128的PP7噬菌体样颗粒,不仅可解决多肽不稳定的问题,也为研究多肽的递送及功能奠定基础。
OBJECTIVE: To obtain PP7 phage-like particles with PPARP fragments (PAP_ (114-128)) on the surface and to confirm that they retain the original biological activity. Methods Genes of PP7 capsid protein single-chain dimer (abbreviated as 2PP7) were amplified by gene mutation and overlap PCR and inserted into vector p ETDuet-1 to obtain plasmid p ETDuet-2PP7 and then obtained by ordinary PCR The plasmid p ETDuet-2PP7-PAP_ (114-128) was inserted into the plasmid p ETDuet-2PP7, which carries the gene encoding PAP_ (114-128) PP7 capsid protein. The recombinant plasmid was transformed into E. coli, The induced product was identified by SDS-PAGE, two-dimensional immunodiffusion and ELISA. Results The 2PP7 gene was obtained by overlapping PCR with ExTaq DNA polymerase. The expression product of the capsid protein single-chain dimer and the wild-type PP7 phage capsid protein had the same antigenicity. The PAP_ (114- 128) epitopes are similar to those of the native human PAP protein and induce higher levels of antibody production. Conclusion The 2PP7 gene containing the repeats can be obtained by combination of gene mutation and overlap PCR. Based on this, the PP7 phage-like particles with PAP114-128 displayed on the surface can not only solve the problem of polypeptide instability, but also Study of peptide delivery and function of the foundation.