P311相互作用蛋白的初步研究

发布时间：2018-03-30 15:46

本文选题：基因　切入点：克隆　出处：《第三军医大学》2005年硕士论文

【摘要】： 深度烧伤创面愈合后常伴随有增生性瘢痕形成,但是对参与增生性瘢痕发生发展的基因及其调控机制目前尚未完全清楚。我们曾经利用基因芯片技术筛选烧伤后早期增生性瘢痕组织与同体正常皮肤组织的差异表达基因研究,找出了97条相关基因。对这些相关基因利用生物信息学一一分析后发现Genebank ID hsu36189的代表基因p311在早期增生性瘢痕组织中呈显著的差异表达,而p311基因的表达在成纤维细胞向肌成纤维细胞转化过程中起着重要作用。那么,该基因的编码蛋白P311在纤维化形成、瘢痕发生发展过程中的作用是什么,其作用靶点是什么?这些问题鲜见文献报道。为此,我们结合蛋白组学研究内容与技术,利用酵母双杂交系统,以融合Gal4 DNA结合区(DNA binding domain, DBD)的P311为诱饵蛋白,筛选了成人肝cDNA文库,以期得到与P311相互作用蛋白的编码基因序列,为进一步的工作奠定基础。主要内容和研究结果如下: 1.诱饵基因的验证p311经PCR扩增、纯化后连接至T载体pTZ57R/T,转化DH5α进行蓝白斑筛选阳性菌落并扩增、抽提质粒测序鉴定p311序列正确。 2.诱饵重组质粒构建与转化以NdeⅠ?BamHⅠ同时双酶切p311 PCR产物和含BD结合域的质粒pGBKT7,回收产物并连接,转化DH5α通过抗性筛选获得阳性细菌克隆,对其进行扩增、提取质粒并利用PCR、酶切和测序鉴定pGBKT7-p311构建成功后,采取醋酸锂法转化感受态酵母菌株AH109,通过缺陷型培养基SD/-Trp筛选阳性克隆,并经PCR再次鉴定正确后保存菌种备用。 3.文库筛选按照Clontech公司的操作手册通过酵母交配实验从预转的成人肝cDNA文库中筛选能够与P311相互作用的阳性克隆。筛选前以pGBKT7空载体作为对照质粒,完成pGBKT7-p311的毒性实验、自激活实验。然后将含有pGBKT7-p311的酵母菌株AH109与含有肝cDNA文库的酵母菌株Y187进行交配后在缺陷型培养基上逐步完成四轮筛选,共获取阳性克隆98个。对98个阳性克隆利用BD、AD载体引物进行PCR鉴定后,得到同时含有诱饵和文库序列的阳性克隆55个。最终从55个阳性克隆中成功提取出40个阳性文库质粒。经转化DH5α、抗性筛选、提取质粒并测序、分类整理后,确认所有阳性克隆均无自激活作用,并与pGBKT7-p311互换宿主
[Abstract]:After the healing of deep burn wound is frequently associated with hypertrophic scar formation, but the gene to participate in the occurrence and development of hypertrophic scar and its regulation mechanism is not yet completely clear. We have been using the difference of gene chip technology to screen early post burn hypertrophic scar and normal skin tissue gene expression studies, we find 97 related genes. One by one analysis of these genes by bioinformatics found on behalf of Genebank ID hsu36189 gene P311 showed a significant difference in the early hypertrophic scar tissues, and the expression of P311 gene in fibroblasts to muscle fiber plays an important role in the cell transformation. Then, the gene encoding protein P311 in fibrosis what is the scar formation, occurrence and development process, what is the target? Reported these problems. Therefore, we combine the egg Study on the content and technology of the white group, using the yeast two hybrid system, the integration of Gal4 and DNA binding domain (DNA binding domain, DBD P311) as the bait protein from the adult liver cDNA library, in order to get the gene encoding a protein interacting with P311, which laid a foundation for further work. The main contents and the results are as follows:
1. the validation of the bait gene. P311 was amplified by PCR and purified to T vector pTZ57R/T. Then DH5 alpha was transformed into blue white spot to screen positive colonies and amplified, and plasmid sequencing was used to identify P311 sequence.
Construction of 2. recombinant bait plasmid and transformed to Nde I? BamH I also digested P311 products of PCR and BD containing plasmid pGBKT7 binding domain, the products recovered and connected, transformed by DH5 alpha positive bacteria clones were screened for amplification, cloning, plasmid was extracted by PCR, enzyme digestion and sequencing pGBKT7-p311 was successfully constructed. After taking plasmid was transformed into competent yeast strain AH109 by defective medium SD/-Trp positive clones were screened by PCR, and again after the identification of strains preservation reserve.
3. library screening Clontech according to the company's operation manual by yeast mating positive clones interacting with P311 can turn from the pre screening experiment of adult liver cDNA library screening. Prior to the pGBKT7 vector as a control plasmid, complete pGBKT7-p311 toxicity test, self activation experiment. Then the pGBKT7-p311 containing yeast strains AH109 and cDNA with liver Library of yeast strain Y187 after mating defective culture gradually completed the four round of screening medium, to obtain a total of 98 positive clones. 98 positive clones using BD and PCR identification of AD vector primers, obtained positive clones containing DNA sequence with the bait and 55. From the end of 55 positive clones the successful extraction of the 40 positive library plasmid. After transformation of DH5 alpha, resistance screening, Plasmid Extraction and sequencing, sorting, confirm that all positive clones showed no self activation and interaction with pGBKT7-p311 Change host

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341

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