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�й����ﻯѧ���������ѧ�� 2005, Vol. 21 Issue (04 ):504-504     DOI:
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IL-10_(23-57)-PE40���ܱ���������ϸ�����
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Soluble Expression,Purification and Cytotoxicity of IL-10_(23-57)-PE40
PENG Qi-Sheng 1),2), ZHANG Guo-Li 1), WU Guang-Mou 1), YUE Yu-Huan 1), MEN Rui-Qi 1), ZHU Ping 1)*( 1)Institute of Veterinary Sciences,Academy of Military Medical Sciences, Changchun 130062, China; 2)College of Veterinary,Jilin University, Changchun 130062, China)

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ժҪ ��IL-10�Ĺ��ܶ���(35��,��IL10��23��57������л�)Ϊ���򲿷���PE40(��ŧ�˾��ⶾ�س�ȥ����������ʣ�ಿ��)�ںϷֱ�����pet20b(+)��pet28a(+)�������鶾��IL102357PE40�����ֱ������,��������pet20b(+)�����鶾����BL21(DE3)pLysS�������ʷ��ڿ�����ʽ���,����pet28a(+)�����鶾����Rosettablue(DE3)���Ը�Ч���ʿ�����ʽ���;����ͨ���������������ˮ�����������ӽ���������ͭ�����׺Ͳ����������ʷ��ڳɷ�,��90%���鶾�ش�Ʒ;ϸ�����ʵ�����,�����鶾��ֻ�Ե��˾���ϸ����ɱ������;ϸ��ELISA��ʾ,�����鶾�ضԵ��˾���ϸ���ɱ������(IC50Ϊ13.9pmolL)�����ŧ�˾��ⶾ�ص����û���.
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�ؼ���� IL-10_(23-57)-PE40   �����Ա��   ����   ϸ����     
Abstract�� An immunotoxin functions by binding to the target cell,becoming internalized,and exerting a toxin action which kills the cell.To develop chimeric protein in which the targeting and toxic portions are combined into a single molecule using recombinant DNA technology,this immunotoxin can target immunocyte populations to prevent organ graft rejection and ameliorate symptoms of autoimmune diseases.The chimeric protein composed of 35 amino acids of interleukin 10(IL-10_ 23-57)fused to truncated form of pseudomonas extoxin(PE) devoid of its native recognition domain,two types of proeukaryonic expression vector,pet-20b-pet-IL-10_ 23-57-PE40 and pet-20a-IL-10_ 23-57-PE40,were constructed, following transfecting into E.coli BL21(DE3)pLys.IL-10_ 23-57-PE40 was expressed in soluble periplasmic and cytoplasmic fraction,respectively. SDS-PAGE showed a single band 43 kD as expected.And Western blot revealed that the expressed protein could specifically bind with anti-IL-10-PE40 antibody.The immunotoxin was further purified to 90% purity with (NH_4)_2SO_4 precipitation,Octy1 Sepharose 4FF,DEAE-Sepharose 4 FF and Cu-based affinity chromatography consecutively.Cytotoxicity assay demonstrated that treatment of mononuclear macrophage carcinoma cell line,RAW264.7,IL-10_ 23-57-PE40 resulted in the cell death with highest efficacy(IC_ 50:13.9 pmol/L),while for bone marrow carcinoma cell line SP2/0 with less efficacy.These results suggest that recombinant immunotoxin IL-10_ 23-57-PE40 targets and kills specific immunocytes and has the potential as therapeutic agent for autoimmune diseases.
Keywords�� IL-10_ 23-57-PE40   soluble expression   purification   cytotoxicity   immunocyte      
Received 2004-10-20;
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����ʤ.IL-10_(23-57)-PE40���ܱ���������ϸ����� [J]  �й����ﻯѧ���������ѧ��, 2005,V21(04 ): 504-504
.Soluble Expression,Purification and Cytotoxicity of IL-10_(23-57)-PE40 [J]  Chinese Journal of Biochemistry and Molecular Biol, 2005,V21(04 ): 504-504
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http://cjbmb.bjmu.edu.cn/CN/     ��     http://cjbmb.bjmu.edu.cn/CN/Y2005/V21/I04 /504