|
|
|
| Expression and Activity of Recombinant Human Glutamate Decarboxylase 65 |
|
|
|
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.
|
|
Received: 31 October 2008
Published: 27 April 2009
|
|
|
|
|
| ［1］ Shi Y，Kanaani J，MenardRose V，et al.Increased expression of GAD65 and GABA in pancreatic betacells impairs firstphase insulin secretion.Am J Physiol Endocrinol Metab，2000，279（3）：E684～694 ［2］ 马明浩，汪洋，金明飞，等.谷氨酸脱羧酶与Ⅰ型糖尿病发病机制.生命的化学，2006，26(1):60～62 Ma M H，Wang Y，Jin M F，et al.Chemistry of Life，2006，26(1):60～62 ［3］ Zimmet P.Antibodies to glutamic acid decarboxylase in the prediction of insulin dependency.Diabetes Res Clin Pract，1996，34：125～131 ［4］ 张颖，郭善一，梁东春，等.大鼠谷氨酸脱羧酶(GAD)毕赤酵母表达重组子的构建.天津医科大学学报，2004，10（1）：43～46 Zhang Y，Guo S Y，Liang D C，et al.Journal of Tianjin Medical University，2004，10（1）：43～46 ［5］ Papouchado M L，Valdez S N，Ghiringhelli D，et al.Expression of properly folded human glutamate decarboxylase 65 as a fusion protein in Escherichia coli.Eur J Biochem，1997，246（2）：350～359 ［6］ Davis K M，Foos T，Bates C S，et al.A novel method for expression and large-scale production of human brain l-glutamate decarboxylase.Biochem Biophys Res Commun，2000，267（3）：777～782 ［7］ 刘芳.血清谷氨酸脱羧酶抗体测定的临床意义.中华内分泌代谢杂志，1997，13（2）：116～118 Liu F.Chin J Endocrinol Metab，1997，13（2）：116～118 ［8］ Rowley M J，Chen Q Y，Teoh K L，et al.Diabetic sera react with the glutamic acid decarboxylase molecule in a dimeric-oligomeric form.Clin Exp Immunol，1996，106（2）：323～328 ［9］ Papouchado M L，Ermácora M R，Poskus E.Detection of glutamic acid decarboxylase （GAD）autoantibodies by indirect immunofluorescence using CHO cells expressing recombinant human GAD65.J Autoimmun，1996，9（5）：689～697 ［10］ Law R H，Rowley M J，Mackay I R，et al.Expression in Saccharomyces cerevisiae of antigenically and enzymatically active recombinant glutamic acid decarboxylase. J Biotechnol，1998，61（1）：57～68 ［11］ 黄静，徐进，左翼，等.重组人胰高血糖素样肽-1表达及生物学活性.中国生物工程杂志，2006，26（1）：50～55 Huang J，Xu J，Zuo Y，et al.China Biotechnology，2006，26（1）：50～55 ［12］ 杨胜远，陆兆新，吕凤霞，等.谷氨酸脱羧酶活力测定中GABA比色定量方法研究.食品科学，2006，26（6）：205～209 Yang S Y，Lu Z X，Lu F X，et al.Food Science，2006，26（6）：205～209 ［13］ 范军，李纯，朱苏文，等.小麦谷氨酸脱羧酶的纯化及部分性质研究.中国生物化学与分子生物学学报，1998，14（5）：641～644 Fan J，Li C，Zhu S W，et al.Chinese Journal of Biochemistry and Molecular Biology，1998，14（5）：641～644 ［14］ Ericson C，Wictorin K，Lundberg C.Ex vivo and in vitro studies of transgene expression in rat astrocytes transduced with lentiviral vectors.Exp Neurol，2002，173（1）：22～30 ［15］ 郁美娟，孟庆华，任吉存，等.基于生命体系中氨基键合标记的荧光探针研究进展.染料与染色，2004，41（3）：137～144 Yu M J，Meng Q H，Ren J C，et al.Dyestuffs and Coloration，2004，41（3）：137～144 |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
