卵黄免疫球蛋白亲和配基分离蛇毒Gln49-PLA_2的研究
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摘要
免疫亲和层析是一种非常有效的生物大分子纯化方法，特别是应用在纯化过程的第一步。本论文根据卵黄免疫球蛋白(IgY)产量大、易于分离等特点，将其作为免疫亲和配基分离相应抗原。
选用长白山白眉蝮蛇蛇毒中的Gln49磷脂酶A_2(Gln49-PLA_2)为抗原免疫产蛋鸡，收集鸡蛋，以天数和ELISA数值(OD值表示IgY相对活性)为坐标绘制免疫滴定曲线，结果表明三次加强免疫后可持续获得抗体，在免疫后51天相对活性达到最高(0.654)，一年后抗体的相对活性维持在0.614，证明特异性抗体的含量至少在一年内基本不变。
几种PEG6000沉淀法粗分IgY的结果显示卵黄液采用PEG6000-乙醇沉淀法，得到的IgY纯度最高，特异活性强，卵磷脂去除较彻底，利于长期保存；用偶联Gln49-PLA_2的Sepharose 4 Fast Flow的亲和介质从卵黄液中一步分离得到特异性抗体，抗体活性提高39倍；以抗GIn49-PLA_2-Sepharose 4 Fast Flow的特异性IgY重复上样，收集相应组分，根据Langmuir-type吸附等温曲线，测定免疫亲和介质的最大载量qm为0.59 mg/mlgel，解离常数K_d为4.4×10~(-5)mg/ml；Western-blot显示抗原与抗体特异性结合。
以抗GIn49-PLA_2特异性IgY为配基偶联到溴化氰活化的Sepharose 4 Fast Flow介质上，一步分离长白山白眉蝮蛇粗毒中的Gln49-PLA_2，所得目的蛋白纯度高达95％，回收率大于83％，产率约为13％，与实验室四步分离Gln49-PLA_2相比，步骤简单，产率提高6.5倍；Anti-Gln49-PLA_2-IgY-Sepharose 4 Fast Flow亲和介质的最大载量qm为0.49mg/ml gel，解离常数K_d为1.66×10~(-7)mg/ml，表明以anti-Gln49-PLA2特异性IgY为配基时不需要苛刻的洗脱条件，是比较理想的生物亲和配基；配基使用60次后，性质仍保持稳定无泄漏情况发生。
以Anti-Gln49-PLA2特异性IgY作为配基纯化大肠杆菌中表达的重组Gln49-PLA2，目的蛋白纯度从4.98％提高到11.5％。
Immunoaffinity chromatography (IAC) is one of the efficient purification methods on biomacromolecule, especially in the first purifying process. Studies on immunoglobulin yolk (IgY) as ligand are carried out, which are based on IgY features that it has a large amount and is easy to isolate.
Hens are immunized using Gln49 phospholipase A2 (Gln49-PLA2) from Agkistrodon blomhoffii ussurensis (Changbai mountain Agkistrodon Halys) snake venom as antigen. Antibody ELISA value at the concentration of 400 ug /ml reaches a peak (0.654) in the 51st day, and has maintained almost as high as 0.614 a year later. Partial purification is conducted according to several kinds of methods of PEG6000. The purity and specific activity is higher by the method of PEG6000, and plenty of phospholipid is got rid of better, which is benefit to store for a long time. Immunoaffinity column (Sepharose 4 Fast Flow) is made with Gln49-PLA2 and used to isolate anti-Gln49-PLA2 antibody from egg yolk. The antibody activity is increased by 39 times. SDS-PAGE and Western-blot analysis confirm that antibody isolated from egg yolk is electrophoretically pure (95%) and specific. By applying various amounts of IgY specific against Gln49-PLA2 to the column, the binding capacity (qm) and dissociation constant (Kd) are 0.59 mg/ml gel and
4.4 X 10-5 mg/ml, respectively, as determined by Langmuir-type adsorption isotherms.
Using specific IgY as ligand, the pure targeted protein (Gln49-PLA2) is obtained, and its purity is just one band on SDS-PAGE. The recovery of Gln49-PLA2 is higher than 83%, and the productivity is about 13%, which is as 6.5 times as Gln49-PLA2by four separating steps. The Kd and qm of the anti-Gln49-PLA2-IgY immunoaffinity column are 1.66X10-7 mg/ml and 0.49 mg/ml gel.
The purity of Gln-49PLA2 expressed in E. coli is increased from 4.98% to 11.5%.
选用长白山白眉蝮蛇蛇毒中的Gln49磷脂酶A_2(Gln49-PLA_2)为抗原免疫产蛋鸡，收集鸡蛋，以天数和ELISA数值(OD值表示IgY相对活性)为坐标绘制免疫滴定曲线，结果表明三次加强免疫后可持续获得抗体，在免疫后51天相对活性达到最高(0.654)，一年后抗体的相对活性维持在0.614，证明特异性抗体的含量至少在一年内基本不变。
几种PEG6000沉淀法粗分IgY的结果显示卵黄液采用PEG6000-乙醇沉淀法，得到的IgY纯度最高，特异活性强，卵磷脂去除较彻底，利于长期保存；用偶联Gln49-PLA_2的Sepharose 4 Fast Flow的亲和介质从卵黄液中一步分离得到特异性抗体，抗体活性提高39倍；以抗GIn49-PLA_2-Sepharose 4 Fast Flow的特异性IgY重复上样，收集相应组分，根据Langmuir-type吸附等温曲线，测定免疫亲和介质的最大载量qm为0.59 mg/mlgel，解离常数K_d为4.4×10~(-5)mg/ml；Western-blot显示抗原与抗体特异性结合。
以抗GIn49-PLA_2特异性IgY为配基偶联到溴化氰活化的Sepharose 4 Fast Flow介质上，一步分离长白山白眉蝮蛇粗毒中的Gln49-PLA_2，所得目的蛋白纯度高达95％，回收率大于83％，产率约为13％，与实验室四步分离Gln49-PLA_2相比，步骤简单，产率提高6.5倍；Anti-Gln49-PLA_2-IgY-Sepharose 4 Fast Flow亲和介质的最大载量qm为0.49mg/ml gel，解离常数K_d为1.66×10~(-7)mg/ml，表明以anti-Gln49-PLA2特异性IgY为配基时不需要苛刻的洗脱条件，是比较理想的生物亲和配基；配基使用60次后，性质仍保持稳定无泄漏情况发生。
以Anti-Gln49-PLA2特异性IgY作为配基纯化大肠杆菌中表达的重组Gln49-PLA2，目的蛋白纯度从4.98％提高到11.5％。
Immunoaffinity chromatography (IAC) is one of the efficient purification methods on biomacromolecule, especially in the first purifying process. Studies on immunoglobulin yolk (IgY) as ligand are carried out, which are based on IgY features that it has a large amount and is easy to isolate.
Hens are immunized using Gln49 phospholipase A2 (Gln49-PLA2) from Agkistrodon blomhoffii ussurensis (Changbai mountain Agkistrodon Halys) snake venom as antigen. Antibody ELISA value at the concentration of 400 ug /ml reaches a peak (0.654) in the 51st day, and has maintained almost as high as 0.614 a year later. Partial purification is conducted according to several kinds of methods of PEG6000. The purity and specific activity is higher by the method of PEG6000, and plenty of phospholipid is got rid of better, which is benefit to store for a long time. Immunoaffinity column (Sepharose 4 Fast Flow) is made with Gln49-PLA2 and used to isolate anti-Gln49-PLA2 antibody from egg yolk. The antibody activity is increased by 39 times. SDS-PAGE and Western-blot analysis confirm that antibody isolated from egg yolk is electrophoretically pure (95%) and specific. By applying various amounts of IgY specific against Gln49-PLA2 to the column, the binding capacity (qm) and dissociation constant (Kd) are 0.59 mg/ml gel and
4.4 X 10-5 mg/ml, respectively, as determined by Langmuir-type adsorption isotherms.
Using specific IgY as ligand, the pure targeted protein (Gln49-PLA2) is obtained, and its purity is just one band on SDS-PAGE. The recovery of Gln49-PLA2 is higher than 83%, and the productivity is about 13%, which is as 6.5 times as Gln49-PLA2by four separating steps. The Kd and qm of the anti-Gln49-PLA2-IgY immunoaffinity column are 1.66X10-7 mg/ml and 0.49 mg/ml gel.
The purity of Gln-49PLA2 expressed in E. coli is increased from 4.98% to 11.5%.
引文
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[2] Amersham Phamacia Biotech Co., Phar macia Biotech Catalog, 1999, 502-629
[3] KTAexplorer 100 System Manual. Amer-sham Pharmacia Biotech Co.,1-8; 63-67
[4] Cheng Y. K., Chase H. S.. Development of operating conditions for protein purification using expanded bed techniques: The effect of the degree of bed expansion on adsorption performance. Biotechnology Bioeng, 1996, 49:512-526
[5] 杨利，贾凌云，邹汉法等．膜色谱技术及其在生化快速分离分析中的应用．生物工程进展，1999，19(1)：48-53
[6] Regnier EE.. Perfusion chromatography. Nature, 1991, 350 ( 18): 634-635
[7] 夏小燕，傅仲华．生物大分子分离纯化技术的重大突破—灌注层析系统．生物工程进展，1994，14(5)：40-46
[8] 熊博晖，王俊德．灌注色谱法的发展和应用．色谱，1997，15(6)：486-489
[9] Margrit Roobol-Bóza, Bertil Andersson. Isolation of Hydrophobic Membrane Proteins by Perfusion Chromatography—Purification of Photosystem Ⅱ Reaction Centers from Spinach Chloroplasts. Anal Biochem, 1996, 235:127-133
[10] 陈冠军．置换层析—重新崛起的生物物质分离技术．生物工程进展，1997，17(3)：44-51
[11] Guhan Jayaraman, Shishir D.Gadam, Steven M. Crarner. Ion-exchange displacement chromatography of proteins: Dextran-based polyelectrolytes as high affinity displacers. J Chromatography, 1993, 630:53-68
[12] Amitava Kundu, Steven M Cramer. Low-Molecular-Weight Displacers for High-Resolution Protein Separations. Anal Biochem, 1997, 248: 111-116
[13] Qing Longchang, Jia Yongchen. Purification of industrial α amylase by reversed micellar extraction. Biotechnology Bioeng, 1995, 48:745-748
[14] 杨青，姚伟，彭明利等．定向合成化学配基亲和层析纯化碱性磷酸酶．生物化学与生物物理学报，1998,30(3)：241
[15] Vladimir I., Muronetz, Timo Korpela. Isolation of antigens and antibodies by affinity chromatography. Journal of chromatography B, 2003, 790:53-66
[16] 邓文涛，陈松林，贺路．草鱼生长激素单抗免疫亲和柱的制备及初步应用．生物化学杂志，1997，13(1)：63
[17] 裴炎，卢晓风，杨星勇等．免疫亲和层析法纯化棕尾别麻蝇(Sarcophagaperegrina)幼虫血淋巴凝集素．中国生物化学与分子生物学报，1999，15(4)：633
[18] 冯小黎，殷剑宁，丛春水等．高效液相亲和层析介质的制备和对基因重组α-干扰素的纯化．中国生化药物杂志，1997，18(1)：5
[19] 谭天伟，桑晋．壳聚糖涂层亲和层析在超氧化物歧化酶纯化中的应用．高校化学工程学报，1998，12 (2)：189
[20] Anshuman V.P., Mohammad M.A.. Multiplex enzyme immuoassay system for the simultaneous detection of multiple allergens in foods. AIChE.J., 1997, 43 ( 12): 3232
[21] Wilchek M.,Miron T.. Quantity fast fraction of a pool of polyclonal antibodies by immunoaffinity membrane chromatography. Reactive &Function Polymers, 1999, 41: 263-268
[22] 廉德君，李林．酵母醇脱氢酶ADHI的纯化及动力学研究．生物化学与生物物理学报，
1996，28(4)：396
[23] 赵翀，王玲，谢蜀生等．Cibacron Blue亲和层析及应用．生物化学杂志，1994，10(5)：621
[24] 金灿煌，范征，赵芳等．人纤溶酶原的亲和色谱分离纯化．中国医药工业杂志，1999，30(2)：49
[25] 王静云，彭孝军，杨冬等．新型染料配基对碱性磷酸酶的亲和纯化．高等学校化学工程学报，2001，15(6)：563-567
[26] Y.Reisner, N.Sharon.. Progress in affinity chromatography. Trends Biochem.Sci., 1980, 5:29
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