两种重组菌中融合蛋白Fv-LDP含量测定方法的比较研究  

Comparison of two kinds of protein determination methods for fusion protein Fv-LDP in the recombinant strain

作　　者：高瑞娟[1] 赵春燕[1] 王玉凤[1] 炼润梅[1] 李电东[1] 

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所,北京100050

出　　处：《中国新药杂志》2010年第4期280-283,331,共5页Chinese Journal of New Drugs

基　　金：重大新药创制重大专项基金(2009ZX09103-136);国家自然科学基金(30400503)

摘　　要：目的：测定抗Ⅳ型胶原酶单链抗体Fv和力达霉素辅基蛋白LDP在大肠杆菌中表达产生的融合蛋白Fv-LDP含量。方法：3瓶培养融合蛋白Fv-LDP表达菌,固定化金属亲和层析纯化融合蛋白Fv-LDP,分步透析使之复性,HPLC测其纯度。比色法测定菌体浓度,SDS-PAGE分离蛋白带,积分密度扫描法测定融合蛋白Fv-LDP表达百分比和含量。结果：在88.7-1 064 mg·L^-1范围内,融合蛋白Fv-LDP浓度和积分密度值呈线性关系（r=0.998 8）,精密度RSD为2.09%（n=8）,平均回收率为95.18%。结论：SDS-PAGE-SpotDenso法操作简单,重复性好,灵敏度高,适用于发酵过程中对融合蛋白Fv-LDP表达产量的控制分析。Objective：To determine the content of fusion protein Fv-LDP produced by Escherichia coli expressing the single-chain Fv antibody against anti-type IV collagenase and lidamycin apoprotein（LDP）.Methods： The recombinant strain expressing fusion protein Fv-LDP was cultured in shaking flask,and then fusion protein Fv-LDP was purified by the immobilized metal affinity chromatography.Next,the purity of fusion protein Fv-LDP was evaluated by HPLC following the renaturation by step-wise dialysis.The colorimetric method was used to set out the concentration of the recombinant strain.After the analysis of the protein bands by SDS-PAGE,the integrated densitometric scanning was performed to determine the expression percentage and the content of fusion protein Fv-LDP.Results： A calibrated linear curve of the concentration of fusion protein Fv-LDP versus the integrated density value was shown within 88.7-1 064 mg·L^-1（r=0.998 8）.The average recovery rate was 95.18% with the RSD of 2.09%（n=8）.Conclusion： This SDS-PAGE-Spot Denso method is more simple,reproducible and sensitive,which can be applied in controlling the production level of fusion protein Fv-LDP during its fermentation process.

关 键 词：单链抗体 力达霉素辅基蛋白 积分密度扫描法 含量 发酵 

分 类 号：R927.2[医药卫生—药学]