重组人谷氨酸脱羧酶65的表达及活性研究

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。

Expression and Activity of Recombinant Human Glutamate Decarboxylase 65

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.