新西兰白兔SOD1基因编码区克隆及生物信息学分析

试验旨在克隆新西兰白兔SOD1基因CDS区并对其进行生物信息学分析。根据GenBank中穴兔SOD 1基因序列(登录号:NM_001082627.2)设计特异性引物,利用RT-PCR技术扩增并克隆新西兰白兔SOD 1基因完整的CDS区序列,测序鉴定后与其他物种进行同源性比对并构建系统进化树,使用SOPMA、SWISS-MODEL、ExPASy、ProtParam等软件和在线工具对蛋白序列进行分析,并预测蛋白分子的亚细胞定位及互作网络。结果显示,新西兰白兔SOD 1基因CDS区长度为459 bp,A、T、C、G碱基含量分别为24.84%、18.95%、23.09%和33.12%,GC含量大于AT含量,编码153个氨基酸。同源性比对结果发现,新西兰白兔与穴兔、卷尾猴、人、黑猩猩、普通狨、骆驼、家犬、猕猴、野猪、小鼠的同源性分别为99.8%、85.2%、85.0%、84.7%、84.1%、83.9%、83.9%、83.7%、83.2%和81.3%。系统进化树结果显示,新西兰白兔和穴兔进化关系最近,并单独聚成一个分支。新西兰白兔SOD1蛋白分子质量为15.7 ku,理论等电点(pI)为5.86,分子式为C672H1084N200O221S5,不稳定指数为23.79,脂肪系数为78.89,平均亲水指数(GRAVY)为-0.276;属水溶性蛋白,无信号肽及跨膜区。SOD1蛋白包含5个潜在的磷酸化修饰位点和3个潜在的糖基化位点;在45-120位氨基酸处存在1个保守的活性中心;二级结构以无规则卷曲为主,占53.59%;三级结构建模显示蛋白易形成同源二聚体。亚细胞定位结果显示,细胞质、细胞核和线粒体分别占65.2%、26.1%和8.7%;SOD1蛋白在体内能与SOD2、PRDX1、PRDX3、PRDX4、DERL1等分子形成互作网络。本试验结果为进一步深入研究SOD 1基因的功能及探索其与相关疾病的关系提供了参考依据。

Induced pluripotent stem cell-derived motor neurons from amyotrophic lateral sclerosis(ALS)patients carrying different superoxide dismutase 1 mutations recapitulate pathological features of ALS

Background:Investigations of the pathogenic mechanisms in motor neurons(MNs)derived from amyotrophic lateral sclerosis(ALS)disease-specific induced pluripotent stem(iPS)cell lines could improve understanding of the issues affecting MNs.Therefore,in this study we explored mutant superoxide dismutase 1(SOD1)protein expression in MNs derived from the iPS cell lines of ALS patients carrying different SOD1 mutations.Methods:We generated induced pluripotent stem cell(iPSC)lines from two familial ALS(FALS)patients withSOD1-V14M andSOD1-C111Y mutations,and then differentiated them into MNs.We investigated levels of the SOD1 protein in iPSCs and MNs,the intracellular Ca2+levels in MNs,and the lactate dehydrogenase(LDH)activity in the process of differentiation into the MNs derived from the controls and ALS patients’iPSCs.Results:The iPSCs from the two FALS patients were capable of differentiation into MNs carrying different SOD1 mutations and differentially expressed MN markers.We detected high SOD1 protein expression and high intracellular calcium levels in both the MN and iPSCs that were derived from the twoSOD1 mutant patients.However,at no time did we observe stronger LDH activity in the patient lines compared with the control lines.Conclusions:MNs derived from patient-specific iPSC lines can recapitulate key aspects of ALS pathogenesis,providing a cell-based disease model to further elucidate disease pathogenesis and explore gene repair coupled with cell-replacement therapy.Incremental mutant expressions of SOD1 in MNs may have disrupted MN function,either causing or contributing to the intracellular calcium disturbances,which could lead to the occurrence and development of the disease.