重组单链抗体-低分子质量尿激酶融合蛋白在CHO细胞中的抗降解研究  

Anti-proteolysis Study of Recombinant Iin-UK Fusion Protein in CHO Cell

作　　者：刘志刚[1] 林建波[1] 杜韫[1] 俞炜源[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物化学与生物物理进展》2005年第6期544-550,共7页Progress In Biochemistry and Biophysics

基　　金：国家高技术'863'计划资助项目(2001AA215351)~~

摘　　要：抗人纤维蛋白单链抗体-低分子质量尿激酶(IIn-UK)融合蛋白,兼有单链抗体对纤维蛋白的亲和性和尿激酶的溶栓活性,有望开发成为新型导向溶栓药物.但基于通用连接肽(G4S)3的IIn-linker-UK融合蛋白在CHO细胞中表达时出现明显的降解.为了解决此问题,利用分子生物学方法,对IIn-UK融合蛋白进行了分子改造,包括置换连接肽,改变两个半分子(moiety)的相对位置,以及对连接肽附近明确的蛋白酶位点进行突变等方法,并分别研究了改造后的11种IIn-linker-UK或UK-linker-IIn突变体在CHO细胞中分泌性表达时的稳定性,最终筛选到一种抗降解的突变体.Fusion protein IIn-UK was constructed by fusing ScFv specific against human fibrin with low molecular urokinase with linker (G4S)3, and this fusion protein was a potential targeting thrombolytic agent. But the fusion protein leaned to be proteolysed while expressed in CHO cells. To overcome this problem, four new linkers were selected from linkers database by using the program at http://ibivu.cs.vu.nl /programs/ linkerdbwww, and four IIn-UK fusion genes were reconstructed by replacing linkers. And other four fusion genes were reconstructed by changing reletive position of two moities and replacing the linker. The degree of proteolysis of eight reconstructed IIn-linker-UK or UK-linker-IIn fusion proteins were analysed with Western blot by using anti-urokinase antibody, the results showed that all eight constructed fusion proteins were degraded partly while expressed in CHO cells. So three new IIn-linker UK fusion genes were constructed by using a new linker coming from pro-urokinase and removing one or two cleavage site of proteolytic enzyme around the linker. The Western blot showed that IIn-UK fusion protein removed two protyolitic enzyme cleavage sites had good anti-proteolysis ability in COS7 cell and CHO cell. Thus it laid a foundation for preparation of IIn-UK fusion protein in CHO cells and further research of targeting thrombolytic agent.

关 键 词：融合蛋白 连接肽 降解 CHO细胞 

分 类 号：Q78[生物学—分子生物学]