Li Jian Feng, Li Jian, Wang Zhi Guo, Liu Hong Zhen, Zhao You Long, Zhang Jin Xi, Zhang Shuang Quan, Liu Jun Ping
Institute of Aging Research, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang Province 311121, China.
The People's No 4 Hospital of Xiaoshan, Hangzhou, Zhejiang Province 311225, China.
Fish Shellfish Immunol. 2015 Feb;42(2):465-72. doi: 10.1016/j.fsi.2014.11.032. Epub 2014 Nov 28.
The interferon-γ-inducible lysosomal thiol reductase (GILT) has been demonstrated to play an important role in the processing and presentation of MHC class II restricted antigen (Ag) by catalyzing disulfide bond reduction. In this study, we cloned a GILT gene homolog from goldfish (designated gGILT), a kind of precious freshwater fish with high market value. The open reading frame of gGILT consists of 756 bases encoding a protein of 251 amino acids with an estimated molecular mass of 27.8 kDa and a theoretical isoelectric point of 5.24. The deduced protein possesses the typical structural features of known GILT proteins, including an active-site motif, a GILT signature sequence, and 10 conserved cysteines. RT-PCR results showed that gGILT and gIFN-γ (goldfish IFN-γ) mRNA were expressed in a tissue-specific manner and obviously up-regulated in splenocytes and the cells from head kidney after induction with LPS. Recombinant gGILT fused with His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Further study revealed that gGILT was capable of catalyzing the reduction of the interchain disulfide bonds from intact IgG. This study shows that gGILT may be involved in the immune response to bacteria challenge and maintain first line of innate immune defense at basal level in goldfish. It also provides the basis for investigating on the role of GILT using goldfish as an animal model.
干扰素γ诱导的溶酶体巯基还原酶（GILT）已被证明通过催化二硫键还原在MHC II类限制性抗原（Ag）的加工和呈递中发挥重要作用。在本研究中，我们从金鱼（命名为gGILT）中克隆了一个GILT基因同源物，金鱼是一种具有高市场价值的珍贵淡水鱼。gGILT的开放阅读框由756个碱基组成，编码一个251个氨基酸的蛋白质，估计分子量为27.8 kDa，理论等电点为5.24。推导的蛋白质具有已知GILT蛋白的典型结构特征，包括一个活性位点基序、一个GILT特征序列和10个保守的半胱氨酸。RT-PCR结果表明，gGILT和gIFN-γ（金鱼IFN-γ）mRNA以组织特异性方式表达，在用LPS诱导后，在脾细胞和头肾细胞中明显上调。与His6标签融合的重组gGILT在大肠杆菌BL21（DE3）中高效表达，并通过Ni-NTA亲和层析纯化。进一步研究表明，gGILT能够催化完整IgG的链间二硫键还原。本研究表明，gGILT可能参与对细菌攻击的免疫反应，并在金鱼的基础水平维持先天免疫防御的第一线。它也为以金鱼为动物模型研究GILT的作用提供了基础。
