人間隙連接蛋白30的互作蛋白的篩選及鑒定
發(fā)布時(shí)間：2018-09-05 18:46
【摘要】： 研究目的: 耳聾是一種常見的感覺障礙性疾病。先天性耳聾在新生兒中的總比例達(dá)到千分之一,另外約一半以上的人到65歲出現(xiàn)明顯的聽力障礙。約50%耳聾是由遺傳因素引起的,70%的遺傳性耳聾不伴有其他癥狀,稱為非綜合征性耳聾。間隙連接蛋白30(CX30或GJB6)是間隙連接蛋白基因家族的一員,CX30突變可導(dǎo)致非綜合征型顯性遺傳性耳聾(DFNA3)和非綜合征型隱性遺傳性耳聾(DFNB1)。此外CX30突變還可導(dǎo)致多汗性外胚層發(fā)育不良、角膜炎-魚鱗病-耳聾綜合征、先天性無毛癥等疾病�？寺《@基因發(fā)現(xiàn)遺傳缺陷是認(rèn)識(shí)遺傳性聾的第一步,進(jìn)一步的耳聾基因功能研究才能認(rèn)識(shí)其在聽覺生理中的作用和致病機(jī)理,以進(jìn)行耳聾的干預(yù)和治療。從2000年蛋白質(zhì)相互作用網(wǎng)絡(luò)圖譜的描繪,蛋白質(zhì)與蛋白質(zhì)之間的相互作用研究成為生命科學(xué)領(lǐng)域的又一個(gè)熱點(diǎn)和里程碑。蛋白質(zhì)與蛋白質(zhì)的相互作用圖譜為蛋白質(zhì)組的功能結(jié)構(gòu)研究提供了一個(gè)重要的框架�；プ鞯鞍椎暮Y選及蛋白之間互作的研究為基因的功能和致病機(jī)理的研究提供了一個(gè)很好的思路。研究表明互作蛋白在間隙連接蛋白的組裝、運(yùn)輸、膜定位、間隙連接通道的形成、間隙連接通信的調(diào)控等一系列過程中均有十分重要的作用。在CX30的功能研究中沒有互作蛋白的研究報(bào)道,因此為研究CX30的功能及可能的致病機(jī)理,進(jìn)行CX30的互作蛋白的研究具有重要的意義。故本研究采用GST-pull down方法篩選CX30互作蛋白和免疫共定位的方法初步鑒定互作蛋白,以探討影響CX30的組裝、運(yùn)輸、膜定位及間隙連接通道形成的可能機(jī)制和可能的致病機(jī)理。 研究方法: (1)構(gòu)建CX30(211-261)-GST原核表達(dá)載體通過primer3網(wǎng)上設(shè)計(jì)引物并引入酶切位點(diǎn),以正常人gDNA為模板,PCR擴(kuò)增CX30的C-末端211.261氨基酸編碼區(qū),目的片段CX30(211.261)插入pGEX-4T-2載體,轉(zhuǎn)化大腸桿菌JM109,抽提質(zhì)粒,雙酶切和測(cè)序證實(shí)插入目的無突變,核對(duì)質(zhì)粒的閱讀框在插入目的基因序列后均無移碼,成功構(gòu)建CX30(211-261)-GST。 (2)親和純化蛋白CX30(211-261)-GST融合原核載體和pGEX-4T-2空載體轉(zhuǎn)化高蛋白表達(dá)的大腸桿菌BL21。IPTG誘導(dǎo)CX30(211-261)-GST融合蛋白和GST小劑量表達(dá),然后誘導(dǎo)大量表達(dá),再以與GST特異結(jié)合的GS4B beads進(jìn)行親和純化CX30(211-261)-GST融合蛋白和GST,純化蛋白以SDS-PAGE膠分離檢測(cè)。 (3)Pull down互作蛋白裂解胎兒腦組織及hela細(xì)胞,提取蛋白質(zhì),先以純化的GST和GS4B bead與提取蛋白質(zhì)孵育進(jìn)行預(yù)吸附,再分別以純化的CX30(211-261)-GST融合蛋白和GST與提取蛋白質(zhì)進(jìn)行孵育,pull down(沉降)可能互作蛋白,SDS-PAGE分離pull down復(fù)合蛋白,比較CX30(211-261)-GST融合蛋白pull down后的復(fù)合蛋白與純化CX30(211-261)-GST融合蛋白和GST pull down后的復(fù)合蛋白之間差異蛋白質(zhì)帶,回收差異蛋白質(zhì)帶膠塊,經(jīng)脫色、修飾和酶解,送Micro Q-TOF質(zhì)譜分析,在NCBInr數(shù)據(jù)庫(kù)檢索篩選出可能的互作蛋白。 (4)免疫共定位鑒定CX30的互作蛋白Keratin16和Tubulin beta-3以正常人DNA為模板,PCR擴(kuò)增CX30 CDS編碼區(qū),構(gòu)建CX30-pCDNA3.1-Myc-His(-)B表達(dá)載體,雙酶切和測(cè)序證實(shí)插入目的無突變及核對(duì)質(zhì)粒的閱讀框在插入目的基因序列后均無移碼。瞬時(shí)轉(zhuǎn)染hela細(xì)胞,進(jìn)行免疫熒光染色,先以熒光顯微鏡上觀察蛋白的表達(dá)及亞細(xì)胞定位情況,再用激光共聚焦顯微鏡觀察免疫共定位情況并掃描和照相。 研究結(jié)果: (1)成功構(gòu)建了CX30(211-261)-GST融合原核載體和CX30-pCDNA3.1-Myc-His(-)B真核表達(dá)載體; (2)大量純化了CX30(211-261)-GST融合蛋白和GST,滿足pull down互作蛋白的需要; (3)在胎兒腦組織,篩選到CX30的4個(gè)可能的互作蛋白:Keratin 16、Camk2b、Tubulin beta-3、alpha-tubulin。在hda細(xì)胞沒有篩選到可能的互作蛋白; (4)CX30與Keratin 16和Tubulin beta-3存在免疫共定位,初步鑒定CX30與Keratin 16和Tubulin beta-3存在相互作用。 研究結(jié)論: (1)GST-pull down是一種行之有效的篩選互作蛋白的方法,本實(shí)驗(yàn)應(yīng)用這個(gè)方法篩選到CX30的4個(gè)可能的互作蛋白:Keratin 16、Camk2b、Tubulin beta-3、alpha-tubulin。Tubulin beta-3和Keratin 16已初步鑒定。 (2)keratin 16、Camk2b、Tubulin beta-3 chain和alpha-tubulin這四個(gè)可能互作蛋白可能在CX30的組裝、運(yùn)輸、間隙連接通道的形成、通道的開關(guān)及間隙連接通信的調(diào)控方面影響CX30的功能。 (3)keratin 16和Camk2b是兩個(gè)新發(fā)現(xiàn)的間隙連接蛋白的互作蛋白。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R764.43;R341
本文編號(hào)：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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R764.43;R341
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