应用酵母双杂交系统初步筛选与SARS-CoV解旋酶相互作用的细胞蛋白
发布时间：2018-09-08 09:16
【摘要】： SARS冠状病毒(Severe Acute Respiratory Syndrome Coronavirus，SARS-CoV)为正链RNA病毒，病毒解旋酶(Helicase，HEL)对SARS-CoV在宿主细胞内复制和增殖是必不可少的。HEL的部分生物学特性已有相关的研究报道，但维持HEL活性的细胞辅助因子、HEL是否具有致病作用等尚未有研究报道。为此，本研究拟采用酵母双杂交系统对大鼠肺上皮细胞文库进行筛选，以期获得与HEL相互作用的蛋白质信息，为研究HEL的作用机制提供线索。 本研究第一部分旨在排除SARS-CoV HEL的自激活作用，为后续的酵母双杂交实验打下基础。为此，，将pGBKT7-HEL(克隆有HEL基因的重组pGBKT7诱饵载体)转化酵母AH109细胞，首先以Western Blot检测融合蛋白，证实HEL蛋白可在AH109细胞中表达，随后通过滤纸法定性检测酵母胞内β半乳糖苷酶活性证明HEL蛋白无自激活作用。 本研究第二部分将pGBKT7-HEL和来源于大鼠eDNA的猎物文库质粒顺序转化酵母AH109细胞，通过营养缺陷培养基进行阳性克隆的挑取。随后将顺序转化所得133个克隆通过三轮营养筛选、酶切鉴定、β-半乳糖苷酶活性检测、一对一酵母双杂交系统的回复性实验确认、测序验证筛除假阳性，最后用Blast软件进行同源性分析研究所筛到的相关蛋白质的信息。通过多重验证，最后确定7种相关蛋白，分别为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)。 总之，本研究初步确定7种与HEL相互作用的大鼠肺上皮细胞蛋白，但这些细胞蛋白的最终确认，以及它们与HEL的相互作用及机制均有待进一步研究。
[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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373.21
【共引文献】
相关期刊论文 前5条
1 李钦云;曹励之;;结核性脑膜炎患儿脑脊液腺苷脱氨酶活力的变化[J];北京医学;2008年03期
2 张e
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