人抗地高辛单链抗体（ADAscFv）的纯化
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摘要
目的 刘德新等人已通过噬菌体抗体库技术绕过免疫成功克隆了人抗地高辛单链抗体(human single-chain variable fragment of anti-Dig antibody, ADAscFv)，并使其在大肠杆菌中得到可溶性表达，目前需依据抗体性质的特点进行分离与纯化。由于不同蛋白质的纯化无通用策略，给制备带来了较大难度和特殊性，本实验的目的即通过试用不同的纯化方法（盐析、阴离子交换、凝胶过滤等），探明适合纯化ADAscFv的最佳纯化方法及条件，为下一步制备诊断Dig中毒的试剂盒和探明ADAscFv对Dig中毒的拮抗作用等后续研究提供实验室依据。
方法 ①培养大肠杆菌(E.coli)HB2151，进行可溶性表达ADAscFv, 离心制备菌液上清，分别经酶联免疫吸附实验（ELISA）和聚丙烯酰氨凝胶电泳（SDS-PAGE）对表达产物进行活性及表达量的检测。②采用不同饱和度硫酸铵分级盐析法（SF）对上述表达物进行初步分离,分别用ELISA和SDS-PAGE法对回收的样品进行活性及纯度的检测，确定硫酸铵的最佳饱和度及适合的温度、沉淀所需时间等条件。③采用HiTrap Q HP(1cm×5cm)强阴离子交换层析柱，通过改变洗脱液的盐浓度、pH值等洗脱条件，对盐析后的样品进行阴离子交换层析法（AEC）纯化, 收集洗脱回收样品用ELISA和SDS-PAGE方法对纯化所得样品进行活性及纯度的检测。④为提高纯化效率，在快速蛋白液相系统（FPLC）对盐析后的样品进行AEC制备级纯化,经不同盐离子(NaCl)浓度的0.05mol/L Tis-HCl溶液梯度洗脱，分别回收各洗脱峰流出样品，用ELISA和SDS-PAGE法对纯化所得样品进行活性及纯度的检测。⑤采用凝胶过滤层析法（GFC），试用不同的纯化条件,对盐析后的样品进行纯化，分别对洗脱峰回收洗脱流出样品，用ELISA和SDS-PAGE对纯化所得样品进行活性及纯度的检测。
结果 ①经ELISA检测显示E.coli HB2151可以稳定性地可溶性表达ADAscFv于菌液培养上清中，SDS-PAGE分析表明菌液上清中仍含大量杂蛋白，需进一步分离纯化。②对不同饱和度硫酸铵盐析后所获样品，分别经ELISA及
SDS-PAGE检测，显示0-50%饱和度硫酸铵盐析所获样品中含有一约36kDa条带，其分子量与预期值(ADAscFv的分子量)相符，且收率高，但杂蛋白也较多。 ③对硫酸铵盐析初步纯化的样品进行阴离子交换层析，回收的样品进行ELISA检测显示洗脱液为0.05mol/L Tris-HCl溶液,pH值9.0，盐离子（NaCl）浓度为0.2mol/L时,纯化所获样品活性高，且SDS-PAGE显示约在36kDa处有一条纯带。 ④在FPLC系统对硫酸铵盐析的样品进行阴离子交换层析，经含NaCl的0.05mol/L Tis-HCl溶液阶梯洗脱，FPLC检测仪显示6个洗脱峰，于第2峰（相当于NaCl浓度为0.2mol/L）收集的样品进行ELISA检测，其活性最高，SDS-PAGE显示分子量约为36kDa的一条纯带，估计回收率至少20%。⑤使用XK16/60空柱，填加基质superdex x75，在FPLC系统进行GFC纯化硫酸铵盐析后的样品，FPLC检测仪显示的流出峰为组峰，且洗脱回收的样品被大量稀释，上样量有限制。
结论 含分泌型ADAscFv的大肠杆菌培养上清经0-50%饱和度硫酸铵盐析粗分离后，在FPLC系统建立阴离子交换层析，选用HiTrap Q HP型强阴离子交换层析柱（1cm(5cm），使用盐离子为NaCl的pH 9.0 0.05 mol/L Tris-HCl溶液阶梯洗脱纯化，于第二峰收集洗脱样品，可获得具有生物活性及高纯度的ADAscFv，该方法操作容易，重现性好，选择性高，所需费用相对低廉，纯化周期短，为快速高效地制备适于临床应用的ADAscFv提供了实验室依据。
Objective To date ,Dexin Liu et al,have obtained a cell line E.coli HB2151 that could secrete human single-chain variable fragment of ADAs (ADAscFv) as soluble protein into culture medium .The ADAscFv can react with Dig and Dig 's analog. Whether ADAscFv serves as measurement of digoxin in plasma following intoxication or clinical treatment, supernatant of the culture medium including soluble ADAscFv should be separated and purified according to it's own features, Nevertheless there is no all-purpose protocol for proteins purification . In order to obtain highly purified ADAscFv and pave the ground for future reseach in which the reagent for diagnosing Dig toxication can be produced and the antagonism of ADAscFv to Dig toxication can be clarified,This work was to explore different methods for separating and purifying supernatant containing soluble ADAscFv.
Methods ①collected supernatant after having cultured E.coli strain HB2151 in culture medium which include soluble ADAscFv, then analysed the antigen-binding activity and purity of ADAscFv respectively with Enzyme-linked immunosorbent assay(ELISA) and Sodium dodecyl sulphate- polyacrylamide gel electrophoresis(SDS-PAGE).②salted-out supernatant with ammonium sulfate of different saturation and analysed the yielded sample respectively with ELISA and SDS-PAGE. ③selected column HiTrap Q HP(1cm×5cm) and eluted it with 0.05mol/L Tris-HCl buffer of different pH, NaCl concentration to purify the salted-out sample through Anion exchange chromatography (AEC),then analysed the purified sample respectively with ELISA and SDS-PAGE.④in order to enhance efficiency, purified the salted-out sample through AEC in Fast performance liquid chromatography (FPLC), analysed the purified sample respectively with ELISA and SDS-PAGE.⑤purified the salted-out sample through gel filtration chromatography (GFC) in FPLC which was performed through using XK16/60 empty column .The column
was filled with matrix superdex x75 and eluted with different eluting fluids and flow rates,etc,analysed the purified elution respectively with ELISA and SDS-PAGE.
Results ①ELISA showed that E.coli strain HB2151 could secrete soluble protein ADAscFv stably,but the cultured supernatant was of many contaminated proteins.②cultured supernatant was seperated through ammonium sulfate gradient-based salting-out,then collected sample to be analysed bioactivity through ELISA and be determined the nature through SDS-PAGE which showed that the supernatant salted-out by ammonium sulfate of 0-50% saturation was of 36kDa lane that accorded with ADAscFv. ③the elution collected through AEC was analysed by ELISA and SDS-PAGE.The result showed that the pH 9.0, 0.05mol/L Tris-HCl buffer containing 0.2mol/L NaCl eluted the column was a comparatively ideal .④Supported by above-step, the column was step-wise washed with pH 9.0, 0.05mol/L Tris-HCl buffer containing NaCl with AEC in FPLC. The elution profile showed six protein peaks and the elution was analysed by ELISA and SDS-PAGE. The presence of the ADAscFv in the fraction corresponded to the second peak. ⑤tried purifying the salted-out sample through Gel filtration chromatography (GFC) in FPLC and observed eluting peaks.Collected elutions respectively and then analysed by ELISA and SDS-PAGE.The eluting peaks was group-peaks.
Discussion In view of our results,we could obtain purified ADAscFv through the following steps which salted-out supernatant contained soluble ADAscFv with 0-50% saturated ammonium sulfate ,then selected column HiTrap Q HP(1cmx5cm) connected with FPLC and AEC step-wise elute the column with pH 9.0,0.05mol/L Tris-HCl (NaCl) buffer .Yielded the second peak elution that contained isolated ADAscFv which has a high purity . Consequently,from this work we can get helpful purification datas and make a good preparation for continued study.
方法 ①培养大肠杆菌(E.coli)HB2151，进行可溶性表达ADAscFv, 离心制备菌液上清，分别经酶联免疫吸附实验（ELISA）和聚丙烯酰氨凝胶电泳（SDS-PAGE）对表达产物进行活性及表达量的检测。②采用不同饱和度硫酸铵分级盐析法（SF）对上述表达物进行初步分离,分别用ELISA和SDS-PAGE法对回收的样品进行活性及纯度的检测，确定硫酸铵的最佳饱和度及适合的温度、沉淀所需时间等条件。③采用HiTrap Q HP(1cm×5cm)强阴离子交换层析柱，通过改变洗脱液的盐浓度、pH值等洗脱条件，对盐析后的样品进行阴离子交换层析法（AEC）纯化, 收集洗脱回收样品用ELISA和SDS-PAGE方法对纯化所得样品进行活性及纯度的检测。④为提高纯化效率，在快速蛋白液相系统（FPLC）对盐析后的样品进行AEC制备级纯化,经不同盐离子(NaCl)浓度的0.05mol/L Tis-HCl溶液梯度洗脱，分别回收各洗脱峰流出样品，用ELISA和SDS-PAGE法对纯化所得样品进行活性及纯度的检测。⑤采用凝胶过滤层析法（GFC），试用不同的纯化条件,对盐析后的样品进行纯化，分别对洗脱峰回收洗脱流出样品，用ELISA和SDS-PAGE对纯化所得样品进行活性及纯度的检测。
结果 ①经ELISA检测显示E.coli HB2151可以稳定性地可溶性表达ADAscFv于菌液培养上清中，SDS-PAGE分析表明菌液上清中仍含大量杂蛋白，需进一步分离纯化。②对不同饱和度硫酸铵盐析后所获样品，分别经ELISA及
SDS-PAGE检测，显示0-50%饱和度硫酸铵盐析所获样品中含有一约36kDa条带，其分子量与预期值(ADAscFv的分子量)相符，且收率高，但杂蛋白也较多。 ③对硫酸铵盐析初步纯化的样品进行阴离子交换层析，回收的样品进行ELISA检测显示洗脱液为0.05mol/L Tris-HCl溶液,pH值9.0，盐离子（NaCl）浓度为0.2mol/L时,纯化所获样品活性高，且SDS-PAGE显示约在36kDa处有一条纯带。 ④在FPLC系统对硫酸铵盐析的样品进行阴离子交换层析，经含NaCl的0.05mol/L Tis-HCl溶液阶梯洗脱，FPLC检测仪显示6个洗脱峰，于第2峰（相当于NaCl浓度为0.2mol/L）收集的样品进行ELISA检测，其活性最高，SDS-PAGE显示分子量约为36kDa的一条纯带，估计回收率至少20%。⑤使用XK16/60空柱，填加基质superdex x75，在FPLC系统进行GFC纯化硫酸铵盐析后的样品，FPLC检测仪显示的流出峰为组峰，且洗脱回收的样品被大量稀释，上样量有限制。
结论 含分泌型ADAscFv的大肠杆菌培养上清经0-50%饱和度硫酸铵盐析粗分离后，在FPLC系统建立阴离子交换层析，选用HiTrap Q HP型强阴离子交换层析柱（1cm(5cm），使用盐离子为NaCl的pH 9.0 0.05 mol/L Tris-HCl溶液阶梯洗脱纯化，于第二峰收集洗脱样品，可获得具有生物活性及高纯度的ADAscFv，该方法操作容易，重现性好，选择性高，所需费用相对低廉，纯化周期短，为快速高效地制备适于临床应用的ADAscFv提供了实验室依据。
Objective To date ,Dexin Liu et al,have obtained a cell line E.coli HB2151 that could secrete human single-chain variable fragment of ADAs (ADAscFv) as soluble protein into culture medium .The ADAscFv can react with Dig and Dig 's analog. Whether ADAscFv serves as measurement of digoxin in plasma following intoxication or clinical treatment, supernatant of the culture medium including soluble ADAscFv should be separated and purified according to it's own features, Nevertheless there is no all-purpose protocol for proteins purification . In order to obtain highly purified ADAscFv and pave the ground for future reseach in which the reagent for diagnosing Dig toxication can be produced and the antagonism of ADAscFv to Dig toxication can be clarified,This work was to explore different methods for separating and purifying supernatant containing soluble ADAscFv.
Methods ①collected supernatant after having cultured E.coli strain HB2151 in culture medium which include soluble ADAscFv, then analysed the antigen-binding activity and purity of ADAscFv respectively with Enzyme-linked immunosorbent assay(ELISA) and Sodium dodecyl sulphate- polyacrylamide gel electrophoresis(SDS-PAGE).②salted-out supernatant with ammonium sulfate of different saturation and analysed the yielded sample respectively with ELISA and SDS-PAGE. ③selected column HiTrap Q HP(1cm×5cm) and eluted it with 0.05mol/L Tris-HCl buffer of different pH, NaCl concentration to purify the salted-out sample through Anion exchange chromatography (AEC),then analysed the purified sample respectively with ELISA and SDS-PAGE.④in order to enhance efficiency, purified the salted-out sample through AEC in Fast performance liquid chromatography (FPLC), analysed the purified sample respectively with ELISA and SDS-PAGE.⑤purified the salted-out sample through gel filtration chromatography (GFC) in FPLC which was performed through using XK16/60 empty column .The column
was filled with matrix superdex x75 and eluted with different eluting fluids and flow rates,etc,analysed the purified elution respectively with ELISA and SDS-PAGE.
Results ①ELISA showed that E.coli strain HB2151 could secrete soluble protein ADAscFv stably,but the cultured supernatant was of many contaminated proteins.②cultured supernatant was seperated through ammonium sulfate gradient-based salting-out,then collected sample to be analysed bioactivity through ELISA and be determined the nature through SDS-PAGE which showed that the supernatant salted-out by ammonium sulfate of 0-50% saturation was of 36kDa lane that accorded with ADAscFv. ③the elution collected through AEC was analysed by ELISA and SDS-PAGE.The result showed that the pH 9.0, 0.05mol/L Tris-HCl buffer containing 0.2mol/L NaCl eluted the column was a comparatively ideal .④Supported by above-step, the column was step-wise washed with pH 9.0, 0.05mol/L Tris-HCl buffer containing NaCl with AEC in FPLC. The elution profile showed six protein peaks and the elution was analysed by ELISA and SDS-PAGE. The presence of the ADAscFv in the fraction corresponded to the second peak. ⑤tried purifying the salted-out sample through Gel filtration chromatography (GFC) in FPLC and observed eluting peaks.Collected elutions respectively and then analysed by ELISA and SDS-PAGE.The eluting peaks was group-peaks.
Discussion In view of our results,we could obtain purified ADAscFv through the following steps which salted-out supernatant contained soluble ADAscFv with 0-50% saturated ammonium sulfate ,then selected column HiTrap Q HP(1cmx5cm) connected with FPLC and AEC step-wise elute the column with pH 9.0,0.05mol/L Tris-HCl (NaCl) buffer .Yielded the second peak elution that contained isolated ADAscFv which has a high purity . Consequently,from this work we can get helpful purification datas and make a good preparation for continued study.
引文
孙光辉,王召军,陈明龙,等.老年人心力衰竭的临床特点和治疗.临床心血管病杂志,1991,7:30-34
Chael R.Ujhelyi,Sylvie Robert.Pharmacokinetic aspects of digoxin-specific Fab therapy in the management of digitalis toxicity.Clin.Pharmacokinet,1995,28(6):483-493
Bowen CA,Krenzelok EP.Clinical application of commonly used contemporary antidotes[J].Drug Saf,1997;16(1):33-34
Smith TW,Bulter VP,Habet E,et al.Determination of theraputic and toxic serum digoxin concentrations by radio-immunoassay.J N Engl J
Med,1969,281:1212-1216
Valdes R Jr,Jortani SA,Gheorghiade M.Standards of laboratory practice:cardiac drug monitoring.National Academy of Clinical Biochemistry.Clin Chem,1998,44:1096-1109
Ball WJ,Kasturi R,Tabet M er al.Isolation and characteriza-tion of human monoclonal antibodies to digoxin.J Immunol,1999, (163):2291-2298
Antman EM,Wenger TL,Butler VP,et al.Treatment of 150 cases of life-threatening digitalis intoxication with digoxin-specific Fab antibody fragments.Circulation 1990,81:1744-1752
Martiny SS,Phelps SJ,Massey KL.treatment of severe digitales intoxication with digoxin-specific antibody fragments:a clinical review.Critical Care Medicine ,1988,16:629-635
叶敏.基因工程研究进展.中华微生物和免疫学杂志,1996,16(4):231-235
刘德新,李小鹰,王琰,等.提高抗地高辛人单链抗体在大肠杆菌分泌型表达的研究.军医进修学院学报,2002,23(1):25-28
赵永芳.生物化学技术原理及应用.北京:科学出版社,2002,101-123
董志伟,王琰.抗体工程.第二版.北京:北京医科大学出版社,2002,333
刘德新,李小鹰,王琰. 单链抗体在大肠杆菌分泌型表达. 军医进修学院学报,2002,23(3):233-235
刘德新,李小鹰,王琰,等.提高抗地高辛人单链抗体在大肠杆菌分泌型表达的研究.军医进修学院学报,2002,23(1):25-28
郭春腾,宁千年,薛伟明,等.硫酸铵盐析在蕲蛇毒凝血酶样酶分离中的应用.福州大学学报,2002,30(1):121-125
李津,俞泳霆,董德祥.生物制药设备和分离纯化技术.北京:化学工业出版社,2003,232-240
黄红辉,刘猛六,胡卓逸.二氢嘧啶酶的分离纯化与性质研究.中国药科大学学报,2000,31(5):389-392
林来兴妹,孟晓静,吉程程,等.角质细胞生长因子2的优化表达及纯化研究.第一军医大学学报,2001,21(12):11-13
Nicole Ameskamp,Christoph Priesner,et al.Pilot scale recovery of
monoclonal antibody by expanded bed ion exchange adsorption. Bioseparation,1999,8:169-188
刘智广,陈志南,刘成刚,等.阴离子交换FPLC制备级纯化单克隆抗体.单克隆抗体通讯,1994,1(1):52-54
彭秀玲,袁汉英.基因工程实验技术.湖南:湖南科学技术出版社,1998,221-222
J.Laroche-Traineau,G.Clofent-Sanchez,X.Santarelli.Three-step purification of bacterially expressed human single-chain Fv antibodies for clinical applications.Journal of chromatography B.2000,737:107-117
Roberto Colangeli,Anna Heijbel,Alam M,et al.Three-step purification of lipopolysaccharide-free,polyhistidine-tagged recombinant antigensof Mycobacterium tuberculosis.Journal of Chromatography B. 1998,714:223-235
陶开华,李越希,汪兴太,等.重组人巨细胞病毒gp52蛋白的纯化及鉴定.中国生物制品学杂志,2001,14(2):75-77
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甄永苏,邵荣光.抗体工程药物.北京:化学工业出版社,2002,95-122
Chael R.Ujhelyi,Sylvie Robert.Pharmacokinetic aspects of digoxin-specific Fab therapy in the management of digitalis toxicity.Clin.Pharmacokinet,1995,28(6):483-493
Bowen CA,Krenzelok EP.Clinical application of commonly used contemporary antidotes[J].Drug Saf,1997;16(1):33-34
Smith TW,Bulter VP,Habet E,et al.Determination of theraputic and toxic serum digoxin concentrations by radio-immunoassay.J N Engl J
Med,1969,281:1212-1216
Valdes R Jr,Jortani SA,Gheorghiade M.Standards of laboratory practice:cardiac drug monitoring.National Academy of Clinical Biochemistry.Clin Chem,1998,44:1096-1109
Ball WJ,Kasturi R,Tabet M er al.Isolation and characteriza-tion of human monoclonal antibodies to digoxin.J Immunol,1999, (163):2291-2298
Antman EM,Wenger TL,Butler VP,et al.Treatment of 150 cases of life-threatening digitalis intoxication with digoxin-specific Fab antibody fragments.Circulation 1990,81:1744-1752
Martiny SS,Phelps SJ,Massey KL.treatment of severe digitales intoxication with digoxin-specific antibody fragments:a clinical review.Critical Care Medicine ,1988,16:629-635
叶敏.基因工程研究进展.中华微生物和免疫学杂志,1996,16(4):231-235
刘德新,李小鹰,王琰,等.提高抗地高辛人单链抗体在大肠杆菌分泌型表达的研究.军医进修学院学报,2002,23(1):25-28
赵永芳.生物化学技术原理及应用.北京:科学出版社,2002,101-123
董志伟,王琰.抗体工程.第二版.北京:北京医科大学出版社,2002,333
刘德新,李小鹰,王琰. 单链抗体在大肠杆菌分泌型表达. 军医进修学院学报,2002,23(3):233-235
刘德新,李小鹰,王琰,等.提高抗地高辛人单链抗体在大肠杆菌分泌型表达的研究.军医进修学院学报,2002,23(1):25-28
郭春腾,宁千年,薛伟明,等.硫酸铵盐析在蕲蛇毒凝血酶样酶分离中的应用.福州大学学报,2002,30(1):121-125
李津,俞泳霆,董德祥.生物制药设备和分离纯化技术.北京:化学工业出版社,2003,232-240
黄红辉,刘猛六,胡卓逸.二氢嘧啶酶的分离纯化与性质研究.中国药科大学学报,2000,31(5):389-392
林来兴妹,孟晓静,吉程程,等.角质细胞生长因子2的优化表达及纯化研究.第一军医大学学报,2001,21(12):11-13
Nicole Ameskamp,Christoph Priesner,et al.Pilot scale recovery of
monoclonal antibody by expanded bed ion exchange adsorption. Bioseparation,1999,8:169-188
刘智广,陈志南,刘成刚,等.阴离子交换FPLC制备级纯化单克隆抗体.单克隆抗体通讯,1994,1(1):52-54
彭秀玲,袁汉英.基因工程实验技术.湖南:湖南科学技术出版社,1998,221-222
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