應(yīng)用酵母雙雜交系統(tǒng)初步篩選與SARS-CoV解旋酶相互作用的細胞蛋白
發(fā)布時間：2018-09-08 09:16
【摘要】： SARS冠狀病毒(Severe Acute Respiratory Syndrome Coronavirus，SARS-CoV)為正鏈RNA病毒，病毒解旋酶(Helicase，HEL)對SARS-CoV在宿主細胞內(nèi)復制和增殖是必不可少的。HEL的部分生物學特性已有相關(guān)的研究報道，但維持HEL活性的細胞輔助因子、HEL是否具有致病作用等尚未有研究報道。為此，本研究擬采用酵母雙雜交系統(tǒng)對大鼠肺上皮細胞文庫進行篩選，以期獲得與HEL相互作用的蛋白質(zhì)信息，為研究HEL的作用機制提供線索。 本研究第一部分旨在排除SARS-CoV HEL的自激活作用，為后續(xù)的酵母雙雜交實驗打下基礎(chǔ)。為此，，將pGBKT7-HEL(克隆有HEL基因的重組pGBKT7誘餌載體)轉(zhuǎn)化酵母AH109細胞，首先以Western Blot檢測融合蛋白，證實HEL蛋白可在AH109細胞中表達，隨后通過濾紙法定性檢測酵母胞內(nèi)β半乳糖苷酶活性證明HEL蛋白無自激活作用。 本研究第二部分將pGBKT7-HEL和來源于大鼠eDNA的獵物文庫質(zhì)粒順序轉(zhuǎn)化酵母AH109細胞，通過營養(yǎng)缺陷培養(yǎng)基進行陽性克隆的挑取。隨后將順序轉(zhuǎn)化所得133個克隆通過三輪營養(yǎng)篩選、酶切鑒定、β-半乳糖苷酶活性檢測、一對一酵母雙雜交系統(tǒng)的回復性實驗確認、測序驗證篩除假陽性，最后用Blast軟件進行同源性分析研究所篩到的相關(guān)蛋白質(zhì)的信息。通過多重驗證，最后確定7種相關(guān)蛋白，分別為Actn2(Actinin alpha 2)、Bcas2(Breast carcinoma amplified sequence 2)、Thap 11(Thr-His-Ala-Pro domain containing 11)、Ada(Adenosine deaminase)、Ddx5(Asp-Glu-Ala-Asp box polypeptide 5)、Dars2(Aspartyl- tRNA synthetase 2)及Smarcb 1(SWI／SNF related，matrix associated，actin dependent regulaor of chromatin，subfamily b，member 1)。 總之，本研究初步確定7種與HEL相互作用的大鼠肺上皮細胞蛋白，但這些細胞蛋白的最終確認，以及它們與HEL的相互作用及機制均有待進一步研究。
[Abstract]:SARS coronavirus (Severe Acute Respiratory Syndrome Coronavirus,SARS-CoV) is a positive strand RNA virus. Viral helicase (Helicase,HEL) is essential to the replication and proliferation of SARS-CoV in host cells. However, it has not been reported whether the cytokine that maintains the activity of HEL has pathogenicity. In order to obtain the protein information of interaction with HEL, this study intends to screen rat lung epithelial cell library by yeast two-hybrid system, and provide clues for studying the mechanism of HEL. The first part of this study was designed to eliminate the self-activation of SARS-CoV HEL and lay a foundation for further yeast two-hybrid experiments. For this reason, pGBKT7-HEL (recombinant pGBKT7 bait vector containing HEL gene) was transformed into yeast AH109 cells. Firstly, the fusion protein was detected by Western Blot, and it was proved that HEL protein could be expressed in AH109 cells. Then the 尾-galactosidase activity of yeast cell was detected qualitatively by filter paper method. It was proved that HEL protein had no self-activation. In the second part of this study, pGBKT7-HEL and the plasmid of prey library derived from rat eDNA were transformed into yeast AH109 cells in sequence, and the positive clones were picked out by nutrient deficiency medium. After that, 133 clones were screened by three rounds of nutrition screening, enzyme digestion, detection of 尾 -galactosidase activity, and confirmed by a one-to-one yeast two-hybrid system. Finally, Blast software was used to analyze the information of related proteins screened by the Institute. Seven related proteins were identified as Actn2 (Actinin alpha 2) Bcas2 (Breast carcinoma amplified sequence 2) Tap11 (Thr-His-Ala-Pro domain containing 11) a (Adenosine deaminase) Ddx5 (Asp-Glu-Ala-Asp box polypeptide 5) Dars2 (Aspartyl- tRNA synthetase 2) and Smarcb 1 (SWI/SNF related,matrix associated,actin dependent regulaor of chromatin,subfamily member 1). In conclusion, seven kinds of pulmonary epithelial cell proteins interacting with HEL were identified in this study, but the final confirmation of these proteins and their interaction with HEL need to be further studied.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R373.21
[Abstract]:SARS coronavirus (Severe Acute Respiratory Syndrome Coronavirus,SARS-CoV) is a positive strand RNA virus. Viral helicase (Helicase,HEL) is essential to the replication and proliferation of SARS-CoV in host cells. However, it has not been reported whether the cytokine that maintains the activity of HEL has pathogenicity. In order to obtain the protein information of interaction with HEL, this study intends to screen rat lung epithelial cell library by yeast two-hybrid system, and provide clues for studying the mechanism of HEL. The first part of this study was designed to eliminate the self-activation of SARS-CoV HEL and lay a foundation for further yeast two-hybrid experiments. For this reason, pGBKT7-HEL (recombinant pGBKT7 bait vector containing HEL gene) was transformed into yeast AH109 cells. Firstly, the fusion protein was detected by Western Blot, and it was proved that HEL protein could be expressed in AH109 cells. Then the 尾-galactosidase activity of yeast cell was detected qualitatively by filter paper method. It was proved that HEL protein had no self-activation. In the second part of this study, pGBKT7-HEL and the plasmid of prey library derived from rat eDNA were transformed into yeast AH109 cells in sequence, and the positive clones were picked out by nutrient deficiency medium. After that, 133 clones were screened by three rounds of nutrition screening, enzyme digestion, detection of 尾 -galactosidase activity, and confirmed by a one-to-one yeast two-hybrid system. Finally, Blast software was used to analyze the information of related proteins screened by the Institute. Seven related proteins were identified as Actn2 (Actinin alpha 2) Bcas2 (Breast carcinoma amplified sequence 2) Tap11 (Thr-His-Ala-Pro domain containing 11) a (Adenosine deaminase) Ddx5 (Asp-Glu-Ala-Asp box polypeptide 5) Dars2 (Aspartyl- tRNA synthetase 2) and Smarcb 1 (SWI/SNF related,matrix associated,actin dependent regulaor of chromatin,subfamily member 1). In conclusion, seven kinds of pulmonary epithelial cell proteins interacting with HEL were identified in this study, but the final confirmation of these proteins and their interaction with HEL need to be further studied.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R373.21
【共引文獻】
相關(guān)期刊論文 前5條
1 李欽云;曹勵之;;結(jié)核性腦膜炎患兒腦脊液腺苷脫氨酶活力的變化[J];北京醫(yī)學;2008年03期
2 張e
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