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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2009, Vol. 29 Issue (04): 12-16    
研究报告     
重组人谷氨酸脱羧酶65的表达及活性研究
汪洋,马明浩,冯震,吴叶林,张绪瑛,金明飞,黄静,吴自荣
 华东师范大学生命科学院
Expression and Activity of Recombinant Human Glutamate Decarboxylase 65
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摘要:

目的:获得有活性的人谷氨酸脱羧酶65(human glutamate decarboxylase 65,hGAD65)用于Ⅰ型糖尿病(type 1 diabetes mellitus,T1DM)检测。方法:将hGAD65基因插入质粒pET32a(+)中,与硫氧还蛋白(thioredox)及六聚组氨酸(hexahistidine)融合,由IPTG诱导融合蛋白(Trx-hGAD65)表达,经两次镍离子亲和层析纯化,通过薄层层析和ELISA研究hGAD65及Trx-hGAD65的活性。结果:在大肠杆菌中实现hGAD65的可溶性表达,经亲和层析得到较纯hGAD65和Trx-hGAD65蛋白,二者具有相同酶学活性,但Trx-hGAD65稳定性更高,并能检测出T1DM患者血清中的hGAD65抗体(hGAD65-Ab)。结论:Trx-hGAD65能在大肠杆菌中可溶性表达,从而为Trx-hGAD65用于T1DM诊断、预防和治疗打下基础。

关键词: Ⅰ型糖尿病;人谷氨酸脱羧酶65;可溶性表达;酶学活性    
Abstract:

Human glutamate decarboxylase 65(hGAD65) is an enzyme that catalyzes the transformation of L-glutamic acid into γ-aminobutyric acid.It has been found that Type 1 diabetes mellitus(T1DM)is an autoimmune disease,in which pancreatic islet β-cells are destroyed due to immune response mediated by autoantigen.hGAD65 is considered as a key autoantigen of the autoimmune response, so anti-hGAD65 antibody(hGAD65-Ab) is the most effective and specific immune marker for T1DM diagnosis, and hGAD65 can be used to detect hGAD65-Ab in serum of T1DM patients.The hGAD65 gene was cloned into pET32a(+),then the recombinant plasmid with hGAD65 was transformed into E.coli BL21(DE3) and expressed by IPTG induction.The fusion protein containing thioredox,hexahistidine and hGAD65 (Trx-hGAD65) was mostly insoluble,but the band of soluble Trx-hGAD65 could also be detected by SDS-PAGE,and it was a great improvement compared with the results reported. Trx-hGAD65 was isolated from lysate and purified by immobilized metal ion affinity chromatography(IMAC).After enterokinase digestion and IMAC purification,hGAD65 with high purity was obtained.Detection of thin-layer chromatography(TLC) showed that both Trx-hGAD65 and hGAD65 had enzymatic activity,whereas Trx-hGAD65 had better stability. Furthermore,it was confirmed that Trx-hGAD65 was able to conjugate with hGAD65-Ab in the serum of T1DM patients by ELISA assay.In conclusion,Trx-hGAD65 instead of hGAD65 can be used for T1DM diagnosis,and its application in prophylaxis and therapy of T1DM is expectable.

Key words: type 1 diabetes mellitus(T1DM);human glutamate decarboxylase 65(hGAD65);soluble expression;enzymatic activity
收稿日期: 2008-10-31 出版日期: 2009-04-27
ZTFLH:  Q786  
基金资助:

上海市科委重大科技项目登山计划(06DZ19016);省部基金

通讯作者: 吴自荣   
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引用本文:

汪洋,马明浩,冯震,吴叶林,张绪瑛,金明飞,黄静,吴自荣. 重组人谷氨酸脱羧酶65的表达及活性研究[J]. 中国生物工程杂志, 2009, 29(04): 12-16.

HONG Xiang- Ma-Meng-Gao- Feng-Shen- Tun-Xie-Lin- Zhang-Xu-Ying- Jin-Meng-Fei- Huang-Jing- Tun-Zi-Rong. Expression and Activity of Recombinant Human Glutamate Decarboxylase 65. China Biotechnology, 2009, 29(04): 12-16.

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https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I04/12

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