应用生物信息学网络资源分析预测融合蛋白的二级结构及其理化性质  

Secondary structure and physico-chemical property of fusion proteins: Analysis with bioinformatics network resource

作　　者：史梦远[1] 王海涛[1] 张芳琳[1] 

机构地区：[1]解放军第四军医大学基础部微生物教研室,陕西省西安市710032

出　　处：《中国组织工程研究与临床康复》2008年第9期1685-1688,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金支持项目(30471626)~~

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,探讨分泌型抗成骨肉瘤单链双功能抗体基因的表达。方法:①将单链抗体基因(ScFv)与白细胞介素2基因亚克隆至反转录病毒表达载体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。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(DNAssist)和蛋白质分析软件(ANTHEPROT V5)分析融合蛋白的氨基酸序列、二级结构及其理化性质。结果:①经酶切及PCR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;Western blotting分析证明ScFv-IL-2基因融合蛋白的表达。②运用DNAssist核酸序列分析软件分析ScFv-IL-2DNA序列翻译并获得了氨基酸序列,运用蛋白质分析软件(ANTHEPROT V5)分析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可以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。AIM： To analyze secondary structure and physico-chemical property of the fusion protein with biointormatics network resource, and explore the expression of a secretory anti-osteoblastic carcinoma single-chain bi-functional antibody gene. METHODS： （1）The single-chain variable fragment （ScFv） antibody gene and interleukin-2 （IL-2） gene were subcloned into corresponding restriction sites of retrovirus expression vector PLxSN. Mediated by liposome, the recombinant plasmid 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 osteosarcoma （OSC） cells were infected by the recombinant retrovirus, terming OSC/ScFv-IL-2. The virus titer was detected by using NIH3T3.（2）The transfected OSC9901 cells by ScFv-IL-2 gene were identified by polymerase chain reaction （PCR）. reversed transcription-PCR and Western blotting. After the fusion protein was constructed, DNAssist and ANTHEPROT V5 softwares were used to analyze the amino acid sequence, the secondary structure, and the physico-chemical property of fusion protein. RESULTS：（1）After the restriction enzyme and PCR identification, the pL（ScFv-IL-2）SN as a fusion protein expression vector, was constructed successfully, and high titer C26 cells were obtained; the expression of recombinant protein was confirmed by Western blotting.（2）On the fusion genes, the DNA sequence was analyzed with DNAssist nucleic acid sequence analysis software, and their secondary structure and physico-chemical property were analyzed with ANTHEPROT V5. CONCLUSION： The property of fusion protein can be analyzed and forecasted by means of bioinformatics network resources, and the approach may provide evidences for investigating single-chain bi-functional antibody gene.

关 键 词：单链抗体 生物信息学资源 融合蛋白ScFv-IL-2基因 生物医学工程 

分 类 号：R318[医药卫生—生物医学工程]