人间隙连接蛋白30的互作蛋白的筛选及鉴定
发布时间：2018-09-05 18:46
【摘要】： 研究目的: 耳聋是一种常见的感觉障碍性疾病。先天性耳聋在新生儿中的总比例达到千分之一,另外约一半以上的人到65岁出现明显的听力障碍。约50%耳聋是由遗传因素引起的,70%的遗传性耳聋不伴有其他症状,称为非综合征性耳聋。间隙连接蛋白30(CX30或GJB6)是间隙连接蛋白基因家族的一员,CX30突变可导致非综合征型显性遗传性耳聋(DFNA3)和非综合征型隐性遗传性耳聋(DFNB1)。此外CX30突变还可导致多汗性外胚层发育不良、角膜炎-鱼鳞病-耳聋综合征、先天性无毛症等疾病。克隆耳聋基因发现遗传缺陷是认识遗传性聋的第一步,进一步的耳聋基因功能研究才能认识其在听觉生理中的作用和致病机理,以进行耳聋的干预和治疗。从2000年蛋白质相互作用网络图谱的描绘,蛋白质与蛋白质之间的相互作用研究成为生命科学领域的又一个热点和里程碑。蛋白质与蛋白质的相互作用图谱为蛋白质组的功能结构研究提供了一个重要的框架。互作蛋白的筛选及蛋白之间互作的研究为基因的功能和致病机理的研究提供了一个很好的思路。研究表明互作蛋白在间隙连接蛋白的组装、运输、膜定位、间隙连接通道的形成、间隙连接通信的调控等一系列过程中均有十分重要的作用。在CX30的功能研究中没有互作蛋白的研究报道,因此为研究CX30的功能及可能的致病机理,进行CX30的互作蛋白的研究具有重要的意义。故本研究采用GST-pull down方法筛选CX30互作蛋白和免疫共定位的方法初步鉴定互作蛋白,以探讨影响CX30的组装、运输、膜定位及间隙连接通道形成的可能机制和可能的致病机理。 研究方法: (1)构建CX30(211-261)-GST原核表达载体通过primer3网上设计引物并引入酶切位点,以正常人gDNA为模板,PCR扩增CX30的C-末端211.261氨基酸编码区,目的片段CX30(211.261)插入pGEX-4T-2载体,转化大肠杆菌JM109,抽提质粒,双酶切和测序证实插入目的无突变,核对质粒的阅读框在插入目的基因序列后均无移码,成功构建CX30(211-261)-GST。 (2)亲和纯化蛋白CX30(211-261)-GST融合原核载体和pGEX-4T-2空载体转化高蛋白表达的大肠杆菌BL21。IPTG诱导CX30(211-261)-GST融合蛋白和GST小剂量表达,然后诱导大量表达,再以与GST特异结合的GS4B beads进行亲和纯化CX30(211-261)-GST融合蛋白和GST,纯化蛋白以SDS-PAGE胶分离检测。 (3)Pull down互作蛋白裂解胎儿脑组织及hela细胞,提取蛋白质,先以纯化的GST和GS4B bead与提取蛋白质孵育进行预吸附,再分别以纯化的CX30(211-261)-GST融合蛋白和GST与提取蛋白质进行孵育,pull down(沉降)可能互作蛋白,SDS-PAGE分离pull down复合蛋白,比较CX30(211-261)-GST融合蛋白pull down后的复合蛋白与纯化CX30(211-261)-GST融合蛋白和GST pull down后的复合蛋白之间差异蛋白质带,回收差异蛋白质带胶块,经脱色、修饰和酶解,送Micro Q-TOF质谱分析,在NCBInr数据库检索筛选出可能的互作蛋白。 (4)免疫共定位鉴定CX30的互作蛋白Keratin16和Tubulin beta-3以正常人DNA为模板,PCR扩增CX30 CDS编码区,构建CX30-pCDNA3.1-Myc-His(-)B表达载体,双酶切和测序证实插入目的无突变及核对质粒的阅读框在插入目的基因序列后均无移码。瞬时转染hela细胞,进行免疫荧光染色,先以荧光显微镜上观察蛋白的表达及亚细胞定位情况,再用激光共聚焦显微镜观察免疫共定位情况并扫描和照相。 研究结果: (1)成功构建了CX30(211-261)-GST融合原核载体和CX30-pCDNA3.1-Myc-His(-)B真核表达载体; (2)大量纯化了CX30(211-261)-GST融合蛋白和GST,满足pull down互作蛋白的需要; (3)在胎儿脑组织,筛选到CX30的4个可能的互作蛋白:Keratin 16、Camk2b、Tubulin beta-3、alpha-tubulin。在hda细胞没有筛选到可能的互作蛋白; (4)CX30与Keratin 16和Tubulin beta-3存在免疫共定位,初步鉴定CX30与Keratin 16和Tubulin beta-3存在相互作用。 研究结论: (1)GST-pull down是一种行之有效的筛选互作蛋白的方法,本实验应用这个方法筛选到CX30的4个可能的互作蛋白:Keratin 16、Camk2b、Tubulin beta-3、alpha-tubulin。Tubulin beta-3和Keratin 16已初步鉴定。 (2)keratin 16、Camk2b、Tubulin beta-3 chain和alpha-tubulin这四个可能互作蛋白可能在CX30的组装、运输、间隙连接通道的形成、通道的开关及间隙连接通信的调控方面影响CX30的功能。 (3)keratin 16和Camk2b是两个新发现的间隙连接蛋白的互作蛋白。
[Abstract]:Research purposes:
Deafness is a common sensory disorder. Congenital deafness accounts for one-thousandth of all newborns, and about half of them develop significant hearing impairment by the age of 65. About 50% of deafness is caused by genetic factors, and 70% of hereditary deafness is not accompanied by other symptoms. It is called non-syndromic deafness. Connexin gap protein CX30 (CX30 or GJB6) is a member of the gap junction protein gene family. CX30 mutations can cause nonsyndromic dominant inherited deafness (DFNA3) and nonsyndromic recessive inherited deafness (DFNB1). The discovery of genetic defects in deafness genes is the first step in understanding hereditary deafness. Further study on the function and pathogenesis of deafness genes in auditory physiology is needed for intervention and treatment of deafness. Interaction maps of proteins and proteins provide an important framework for the study of functional structure of proteomics. Screening of interacting proteins and the study of protein-protein interactions provide a good idea for the study of gene function and pathogenesis. Protein plays an important role in the assembly, transportation, membrane localization, gap junction channel formation, gap junction communication regulation and so on. There is no report on the interaction protein in the functional study of CX30. Therefore, in order to study the function and possible pathogenesis of CX30, the interaction protein of CX30 is carried out. Therefore, GST-pull down method was used to screen CX30 interacting proteins and identify the interacting proteins preliminarily in order to explore the possible mechanism and pathogenesis of the assembly, transport, membrane localization and gap junction channel formation of CX30.
Research methods:
(1) CX30 (211-261) - GST prokaryotic expression vector was constructed and primer3 primers were designed and digested. The C-terminal 211.261 amino acid coding region of CX30 was amplified by PCR using normal human gDNA as template. The target fragment CX30 (211.261) was inserted into pGEX-4T-2 vector, transformed into E.coli JM109, and plasmid was extracted. Double digestion and sequencing confirmed that the inserted target fragment was neutral. CX30 (211-261) -GST was successfully constructed by inserting the target gene sequence into the reading frame of the plasmid.
(2) The fusion prokaryotic vector of CX30 (211-261) -GST fusion protein and pGEX-4T-2 empty vector transformed high protein expression E.coli BL21. IPTG induced low-dose expression of CX30 (211-261) -GST fusion protein and GST, then induced large-scale expression, and then affinity purified CX30 (211-261) -GST fusion protein and GST by GS4B beads specifically binding to GST. The protein was separated by SDS-PAGE gel.
(3) Pull down interacting protein cleaves fetal brain tissue and HeLa cells, extracts protein, first with purified GST and GS4B bead and extracts protein incubation for pre-adsorption, then with purified CX30 (211-261) -GST fusion protein and GST and extracts protein respectively for incubation, pull down (sedimentation) may interact with protein, SDS-PAGE separation pull down composite. Comparing the different protein bands between CX30 (211-261) - GST fusion protein pull down and purified CX30 (211-261) - GST fusion protein and GST pull down, recovering the different protein bands, decoloring, modifying and enzymatic hydrolysis, sending micro-Q-TOF mass spectrometry analysis to screen out possible interactions in NCBInr database. Protein.
(4) CX30 interacting proteins Keratin 16 and Tubulin beta-3 were identified by co-immunolocalization. CX30-pCDNA3.1-Myc-His (-) B expression vector was constructed by PCR amplification of CX30-CDS coding region using normal human DNA as template. Double enzyme digestion and sequencing confirmed that the inserted target-free and the reading frame of the nucleotide plasmid did not shift after inserting the target gene sequence. Immunofluorescence staining was used to observe the protein expression and subcellular localization. Laser confocal microscopy was used to observe the immunolocalization and scanning and photography.
Research findings:
(1) successfully constructed CX30 (211-261) -GST fusion prokaryotic vector and CX30-pCDNA3.1-Myc-His (-) B eukaryotic expression vector.
(2) a large number of CX30 (211-261) -GST fusion proteins and GST were purified to meet the needs of pull down interacting protein.
(3) In fetal brain tissues, four possible CX30 interacting proteins were screened: Keratin 16, Camk2b, Tubulin beta-3, alpha-tubulin. No possible interacting proteins were found in HDA cells.
(4) CX30 was co-localized with Keratin 16 and Tubulin beta-3, and the interaction between CX30 and Keratin 16 and Tubulin beta-3 was preliminarily identified.
Research conclusions:
(1) GST-pull down is an effective method for screening interacting proteins. Four possible CX30 interacting proteins, Keratin 16, Camk2b, Tubulin beta-3, alpha-tubulin.Tubulin beta-3 and Keratin 16, have been identified by this method.
(2) The four possible interacting proteins keratin 16, Camk2b, Tubulin beta-3 chain and alpha-tubulin may affect the function of CX30 in assembly, transportation, gap junction channel formation, channel switching and gap junction communication regulation.
(3) keratin 16 and Camk2b are two new interacting proteins of gap junction proteins.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R764.43;R341
本文编号：2225141
[Abstract]:Research purposes:
Deafness is a common sensory disorder. Congenital deafness accounts for one-thousandth of all newborns, and about half of them develop significant hearing impairment by the age of 65. About 50% of deafness is caused by genetic factors, and 70% of hereditary deafness is not accompanied by other symptoms. It is called non-syndromic deafness. Connexin gap protein CX30 (CX30 or GJB6) is a member of the gap junction protein gene family. CX30 mutations can cause nonsyndromic dominant inherited deafness (DFNA3) and nonsyndromic recessive inherited deafness (DFNB1). The discovery of genetic defects in deafness genes is the first step in understanding hereditary deafness. Further study on the function and pathogenesis of deafness genes in auditory physiology is needed for intervention and treatment of deafness. Interaction maps of proteins and proteins provide an important framework for the study of functional structure of proteomics. Screening of interacting proteins and the study of protein-protein interactions provide a good idea for the study of gene function and pathogenesis. Protein plays an important role in the assembly, transportation, membrane localization, gap junction channel formation, gap junction communication regulation and so on. There is no report on the interaction protein in the functional study of CX30. Therefore, in order to study the function and possible pathogenesis of CX30, the interaction protein of CX30 is carried out. Therefore, GST-pull down method was used to screen CX30 interacting proteins and identify the interacting proteins preliminarily in order to explore the possible mechanism and pathogenesis of the assembly, transport, membrane localization and gap junction channel formation of CX30.
Research methods:
(1) CX30 (211-261) - GST prokaryotic expression vector was constructed and primer3 primers were designed and digested. The C-terminal 211.261 amino acid coding region of CX30 was amplified by PCR using normal human gDNA as template. The target fragment CX30 (211.261) was inserted into pGEX-4T-2 vector, transformed into E.coli JM109, and plasmid was extracted. Double digestion and sequencing confirmed that the inserted target fragment was neutral. CX30 (211-261) -GST was successfully constructed by inserting the target gene sequence into the reading frame of the plasmid.
(2) The fusion prokaryotic vector of CX30 (211-261) -GST fusion protein and pGEX-4T-2 empty vector transformed high protein expression E.coli BL21. IPTG induced low-dose expression of CX30 (211-261) -GST fusion protein and GST, then induced large-scale expression, and then affinity purified CX30 (211-261) -GST fusion protein and GST by GS4B beads specifically binding to GST. The protein was separated by SDS-PAGE gel.
(3) Pull down interacting protein cleaves fetal brain tissue and HeLa cells, extracts protein, first with purified GST and GS4B bead and extracts protein incubation for pre-adsorption, then with purified CX30 (211-261) -GST fusion protein and GST and extracts protein respectively for incubation, pull down (sedimentation) may interact with protein, SDS-PAGE separation pull down composite. Comparing the different protein bands between CX30 (211-261) - GST fusion protein pull down and purified CX30 (211-261) - GST fusion protein and GST pull down, recovering the different protein bands, decoloring, modifying and enzymatic hydrolysis, sending micro-Q-TOF mass spectrometry analysis to screen out possible interactions in NCBInr database. Protein.
(4) CX30 interacting proteins Keratin 16 and Tubulin beta-3 were identified by co-immunolocalization. CX30-pCDNA3.1-Myc-His (-) B expression vector was constructed by PCR amplification of CX30-CDS coding region using normal human DNA as template. Double enzyme digestion and sequencing confirmed that the inserted target-free and the reading frame of the nucleotide plasmid did not shift after inserting the target gene sequence. Immunofluorescence staining was used to observe the protein expression and subcellular localization. Laser confocal microscopy was used to observe the immunolocalization and scanning and photography.
Research findings:
(1) successfully constructed CX30 (211-261) -GST fusion prokaryotic vector and CX30-pCDNA3.1-Myc-His (-) B eukaryotic expression vector.
(2) a large number of CX30 (211-261) -GST fusion proteins and GST were purified to meet the needs of pull down interacting protein.
(3) In fetal brain tissues, four possible CX30 interacting proteins were screened: Keratin 16, Camk2b, Tubulin beta-3, alpha-tubulin. No possible interacting proteins were found in HDA cells.
(4) CX30 was co-localized with Keratin 16 and Tubulin beta-3, and the interaction between CX30 and Keratin 16 and Tubulin beta-3 was preliminarily identified.
Research conclusions:
(1) GST-pull down is an effective method for screening interacting proteins. Four possible CX30 interacting proteins, Keratin 16, Camk2b, Tubulin beta-3, alpha-tubulin.Tubulin beta-3 and Keratin 16, have been identified by this method.
(2) The four possible interacting proteins keratin 16, Camk2b, Tubulin beta-3 chain and alpha-tubulin may affect the function of CX30 in assembly, transportation, gap junction channel formation, channel switching and gap junction communication regulation.
(3) keratin 16 and Camk2b are two new interacting proteins of gap junction proteins.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R764.43;R341
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