摘要
目的实现抗Ⅳ型胶原酶单链抗体(Fv)与lidamycin辅基蛋白(LDP)结合构成的融合蛋白Fv-LDP产生菌的高密度发酵,并测定融合蛋白Fv-LDP免疫活性。方法先在5L发酵罐中进行工程菌分批发酵和补料分批发酵,再用固定化金属亲和层析从菌体中纯化融合蛋白Fv-LDP,分步透析使之复性,酶联免疫吸附试验和免疫荧光细胞化学染色检测其免疫活性。结果确定分阶段恒速补料分批发酵为工程菌最佳高密度发酵模式,融合蛋白Fv-LDP最高表达量为4g/L,A600为55左右,培养时间约14h。融合蛋白Fv-LDP对Ⅳ型胶原酶呈阳性免疫反应,能与PG细胞特异结合。结论确定了工程菌高密度发酵工艺,融合蛋白Fv-LDP免疫活性良好,为融合蛋白Fv-LDP与lidamycin烯二炔发色团(AE)构成的小型高效化抗体药物Fv-LDP-AE的研究奠定良好基础。
Objective To accomplish high cell-density fermentation of the engineered strain producing fusion protein Fv-LDP consisting of single-chain Fv antibody directed against type IV collagenase （Fv） and lidamycin apoprotein （LDP）, and to determine the immunological activity of the fusion protein Fv-LDP. Methods The batch and fed-batch fermentation were first conducted in 5-liter fermentation jar. Then, fusion protein Fv-LDP was purified from the engineered strains and renaturized by the immobilized metal affinity chromatography and stepwise dialysis, respectively. Finally, the immunological activity of fusion protein Fv-LDP was analyzed by enzyme-linked immunosorbent assay and immunofluorescent cytochemical staining. Results The stepwise and constant speed fed-batch fermentation was the optimum model for high cell-density fermentation of the engineered strain with the highest yield of fusion protein Fv-LDP of 4g/L and A600 of 55 or so in around 14 h. Fusion protein Fv-LDP showed positive immunoreactivity against type Ⅳ collagenase and could specifically bind to PG cells. Conclusions The stepwise and constant speed fed-batch fermentation process of the engineered strain was determined and the fusion protein Fv-LDP showed good immunological activity to type Ⅳ collagenase. This will provide the good basis for the study of antibody drug Fv-LDP-AE with downsized molecule and highly potent antitumor efficacy which consists of fusion protein Fv-LDP and the active enediyne （AE） of lidamyein.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2009年第9期536-540,共5页
Chinese Journal of Antibiotics
基金
<重大新药创制>重大科技专项资金(2009ZX09103-136)
国家自然科学基金(No.30400543)
