摘要
目的利用生物信息学网络资源分析融合蛋白的二级结构及其理化性质,并探讨分泌型抗成骨肉瘤单链双功能抗体基因的表达。方法采用聚合酶链式反应(PCR)将人工合成的抗体分泌信号肽序列加在抗成骨肉瘤单链抗体(scFv)基因5′端,其3′与白介素-2(IL-2)基因连接构成分泌型单链双功能抗体scFv-IL-2基因,将该基因克隆至逆转录病毒表达载体PLxSN,重组质粒pL(scFv-IL-2)SN在脂质体介导下转染PA317包装细胞,G418筛选,直至出现抗性克隆,扩大培养,用NIH3T3测定病毒滴度,将重组病毒感染人成骨肉瘤细胞命名为OSC/scFv-IL-2,以PCR,RT-PCR以及Western blotting对scFv-IL-2基因修饰的OSC9901细胞进行鉴定。在构建融合蛋白之后,运用DNA分析软件DNAs-sist和蛋白质分析软件ANTHEPROTV5分析融合蛋白的氨基酸序列、二级结构及其理化性质。结果经酶切、PCR及Western blotting分析鉴定,成功地构建了融合基因表达载体pL(scFv-IL-2)SN,并获得高滴度产毒细胞株C26,scFv-IL-2融合蛋白通过DNAssist和ANTHEPROTV5软件分析获得了融合蛋白的二级结构及其理化性质。结论利用生物信息学网络资源进行分析预测融合蛋白的性质,为进一步探讨单链双功能抗体基因融合蛋白提供依据。
Objective To analyze secondary structure and the physico-chemical property of the fusion protein with software, and to investigate the expression of single-chain bifunctional antibody gene of secreting type anti-osteogenic sarcoma. Methods By the technology of polymerase chain reaction（PCR） , the sequenced genes of secretary signal peptide of artificial synthetical antibody were added to single-chain antibody（scFv） genes ＇5＇ of anti-osteogenic sarcoma and the interleukin-2 （IL-2） genes were added to scFv- IL-2 genes, the sequenced genes were subcloned into corresponding restriction sites of PLxSN in turn to generate coexpression vector. The pL（scFv-IL-2） SN was packaged with PA317 and selected in G418 to obtain the positive clones, which were able to produce stable retrovirus,and then OSC9901 cells were infected by the recombinant retrovirus. The positive clones were obtained after G418 selection and were termed OSC/scFv-IL-2, and fusion protein were identified by Western bloting in the transfected OSC9901 cells. In the works, DNAssist and ANTHEPROT V5 were used to analyze the sequence ,the secondary structure, and the physical and chymic character. Results The inserted fragments in all constructed plasmids were structurally confirmed to be consistent with that of the published data. Similarly ,the G418 selected vectors PL（scFv-IL-2） SN was confirmed to be successful in the stable expression of the objective proteins in the target cells, and expression of recombinant protein was confirmed by Western blotting. On the fusion genes, the obtained sequence was analyzed with DNAssist and ANTHEPROT VS. Conclusion The softwares which are downloaded from web should be fully used to analyze biologic information, and the approach may have application in a gene therapy context.
出处
《新乡医学院学报》
CAS
2008年第2期134-137,共4页
Journal of Xinxiang Medical University
关键词
单链抗体
生物信息学资源
融合蛋白
single-chain bi-functional antibody
bioinformatical resource
fusion protein
