胞浆接头蛋白Dok5的表达及生物信息学分析  

Expression and bioinformatics analysis of downstream of tyrosine kinase 5

作　　者：庞云丽 杨晶 杨云 张萌 赵思雯 鲁帅尧[1] PANG Yun-li;YANG Jing;YANG Yun;ZHANG Meng;ZHAO Si-wen;LU Shuai-yao(Institute of Medical Biology,Peking Union Medical College,Chinese Academy of Medical Sciences,Kunming 650118,China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650118

出　　处：《生物技术》2020年第2期121-128,共8页Biotechnology

基　　金：云南省中青年学术和技术带头人后备人才培养基金项目(2017HB068);云南省卫生和计划生育委员会医学学科带头人培养计划(D-201653)。

摘　　要：[目的]构建携带小鼠Dok5基因及其PH、PTB结构域的原核表达载体,并表达对应GST融合蛋白。[方法]从小鼠脑组织提取总RNA,逆转录后经PCR扩增酶切、连接及转化大肠杆菌DH5a,构建携带Dok5全长(FL),PTB、PH结构域的原核表达质粒,经酶切及测序鉴定之后,转化大肠杆菌BL21,经IPTG诱导表达蛋白,再利用Clutathion Sepha-rose 4B亲和层析柱纯化得到融合蛋白,并通过Western Botting检测其与相应抗体的反应性。利用生物信息学相关软件(ClustalW2、ProtParam、ProtScale、NetPhos 2.0 Server、SOPMA.Swissmodel)预测Dok5蛋白的理化性质和磷酸化位点以及二、三级结构。[结果]构建了小鼠Dok5 FL、PH.PTB的原核表达质粒,并得到了浓度分别为0.3 mg/ml.0.6 mg/mL、1 mg/mL的pGEX 6p-1-Dok5 FL、PH、PTB蛋白,其中Dok5 FL融合蛋白与相应抗体具有良好的反应性。Dok5长度为921 bp,编码306个氨基酸,在人、鼠、猴中高度保守。Dok5相对分子质量为35.453 kDa,理论pl为9.0,包含50个潜在磷酸化位点。[结论]分别得到了63 kDa.39 kDa、37 kDa的Dok5 FL、PH、PTB的GST融合蛋白,预测Dok5是--种亲水性的不稳定蛋白。[Objective]To construct prokaryotic expression vector carrying mouse Dok5 and its PH and PTB domains,and to express GST fusion protein.[Method]The total RNA was extracted from mouse brain tissue,amplfied by PCR,digested,linked and transformed into E.coli DH5a.The prokaryotic expression plasmid carrying Dok5 full-length(FL)or PTB or PH domain was constructed.After the identification of digestion and sequencing,E.coli BL21 was transformed and induced by IPTG to make fusion protein,and then the fusion protein was purified by glutathione Sepharose 4 B afinity chromatography,and its reac-tivity with the corresponding antibody was detected by Western Blotting.Bioinformatics software(clustalw2,protparam,Protscale,netphos 2.0 server,sopma,swissmodel)was used to predict the physical and chemical properties,phosphorylation sites and secondary and tertiary struetures of Dok5.[Result]The prokaryotic expression plasmids of Dok5 FL(306aa),PH do-main(1-113aa)and PTB domain(114-220aa)were constructed,and pGEX 6p-1-Dok5 FL,PH and PTB proteins with concentrations of 0.3 mg/ml,0.6 mg mL and 1 mg/mL,respectively,were obtained.Dok5 includes 921 bp and encodes 306 a mino acids.It is highly conserved in human,mouse and Rhesus.Its relative molecular weight is 35.453 kDa and theoretical PI is9.01.It is a kind of hydrophilie unstable protein.[Concelusion]The GST fusion proteins of Dok5 FL,PH and PTB were ob-tained at 63 kDa,39 kDa and 37 kDa,respectively.

关 键 词：DOK5 原核表达 理化性质 磷酸化位点 蛋白结构 

分 类 号：Q786[生物学—分子生物学]