【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(duì)SARS-CoV在宿主細(xì)胞內(nèi)復(fù)制和增殖是必不可少的。HEL的部分生物學(xué)特性已有相關(guān)的研究報(bào)道，但維持HEL活性的細(xì)胞輔助因子、HEL是否具有致病作用等尚未有研究報(bào)道。為此，本研究擬采用酵母雙雜交系統(tǒng)對(duì)大鼠肺上皮細(xì)胞文庫(kù)進(jìn)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(jī)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(shí)驗(yàn)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(xì)胞，首先以Western Blot檢測(cè)融合蛋白，證實(shí)HEL蛋白可在AH109細(xì)胞中表達(dá)，隨后通過(guò)濾紙法定性檢測(cè)酵母胞內(nèi)β半乳糖苷酶活性證明HEL蛋白無(wú)自激活作用。 本研究第二部分將pGBKT7-HEL和來(lái)源于大鼠eDNA的獵物文庫(kù)質(zhì)粒順序轉(zhuǎn)化酵母AH109細(xì)胞，通過(guò)營(yíng)養(yǎng)缺陷培養(yǎng)基進(jìn)行陽(yáng)性克隆的挑取。隨后將順序轉(zhuǎn)化所得133個(gè)克隆通過(guò)三輪營(yíng)養(yǎng)篩選、酶切鑒定、β-半乳糖苷酶活性檢測(cè)、一對(duì)一酵母雙雜交系統(tǒng)的回復(fù)性實(shí)驗(yàn)確認(rèn)、測(cè)序驗(yàn)證篩除假陽(yáng)性，最后用Blast軟件進(jìn)行同源性分析研究所篩到的相關(guān)蛋白質(zhì)的信息。通過(guò)多重驗(yàn)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(xì)胞蛋白，但這些細(xì)胞蛋白的最終確認(rèn)，以及它們與HEL的相互作用及機(jī)制均有待進(jìn)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(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R373.21

【共引文獻(xiàn)】

相關(guān)期刊論文 前5條

1 李欽云;曹勵(lì)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(xué);2008年03期

2 張e

本文編號(hào)：2230098