本文選題：基因　切入點(diǎn)：克隆　出處：《第三軍醫(yī)大學(xué)》2005年碩士論文

【摘要】： 深度燒傷創(chuàng)面愈合后常伴隨有增生性瘢痕形成,但是對參與增生性瘢痕發(fā)生發(fā)展的基因及其調(diào)控機(jī)制目前尚未完全清楚。我們曾經(jīng)利用基因芯片技術(shù)篩選燒傷后早期增生性瘢痕組織與同體正常皮膚組織的差異表達(dá)基因研究,找出了97條相關(guān)基因。對這些相關(guān)基因利用生物信息學(xué)一一分析后發(fā)現(xiàn)Genebank ID hsu36189的代表基因p311在早期增生性瘢痕組織中呈顯著的差異表達(dá),而p311基因的表達(dá)在成纖維細(xì)胞向肌成纖維細(xì)胞轉(zhuǎn)化過程中起著重要作用。那么,該基因的編碼蛋白P311在纖維化形成、瘢痕發(fā)生發(fā)展過程中的作用是什么,其作用靶點(diǎn)是什么?這些問題鮮見文獻(xiàn)報道。為此,我們結(jié)合蛋白組學(xué)研究內(nèi)容與技術(shù),利用酵母雙雜交系統(tǒng),以融合Gal4 DNA結(jié)合區(qū)(DNA binding domain, DBD)的P311為誘餌蛋白,篩選了成人肝cDNA文庫,以期得到與P311相互作用蛋白的編碼基因序列,為進(jìn)一步的工作奠定基礎(chǔ)。主要內(nèi)容和研究結(jié)果如下: 1.誘餌基因的驗(yàn)證p311經(jīng)PCR擴(kuò)增、純化后連接至T載體pTZ57R/T,轉(zhuǎn)化DH5α進(jìn)行藍(lán)白斑篩選陽性菌落并擴(kuò)增、抽提質(zhì)粒測序鑒定p311序列正確。 2.誘餌重組質(zhì)粒構(gòu)建與轉(zhuǎn)化以NdeⅠ?BamHⅠ同時雙酶切p311 PCR產(chǎn)物和含BD結(jié)合域的質(zhì)粒pGBKT7,回收產(chǎn)物并連接,轉(zhuǎn)化DH5α通過抗性篩選獲得陽性細(xì)菌克隆,對其進(jìn)行擴(kuò)增、提取質(zhì)粒并利用PCR、酶切和測序鑒定pGBKT7-p311構(gòu)建成功后,采取醋酸鋰法轉(zhuǎn)化感受態(tài)酵母菌株AH109,通過缺陷型培養(yǎng)基SD/-Trp篩選陽性克隆,并經(jīng)PCR再次鑒定正確后保存菌種備用。 3.文庫篩選按照Clontech公司的操作手冊通過酵母交配實(shí)驗(yàn)從預(yù)轉(zhuǎn)的成人肝cDNA文庫中篩選能夠與P311相互作用的陽性克隆。篩選前以pGBKT7空載體作為對照質(zhì)粒,完成pGBKT7-p311的毒性實(shí)驗(yàn)、自激活實(shí)驗(yàn)。然后將含有pGBKT7-p311的酵母菌株AH109與含有肝cDNA文庫的酵母菌株Y187進(jìn)行交配后在缺陷型培養(yǎng)基上逐步完成四輪篩選,共獲取陽性克隆98個。對98個陽性克隆利用BD、AD載體引物進(jìn)行PCR鑒定后,得到同時含有誘餌和文庫序列的陽性克隆55個。最終從55個陽性克隆中成功提取出40個陽性文庫質(zhì)粒。經(jīng)轉(zhuǎn)化DH5α、抗性篩選、提取質(zhì)粒并測序、分類整理后,確認(rèn)所有陽性克隆均無自激活作用,并與pGBKT7-p311互換宿主
[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

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 田云,盧向陽;蛋白質(zhì)間相互作用研究技術(shù)進(jìn)展[J];生物學(xué)通報;2003年05期

2 朱靜儀;;酵母雙雜交技術(shù)的發(fā)展[J];中山大學(xué)研究生學(xué)刊(自然科學(xué)、醫(yī)學(xué)版);2002年02期

3 馬兵,吳軍,易紹萱,羅高興,賀偉峰,王珍祥,陳烯偉;表達(dá)譜基因芯片篩選燒傷后增生性瘢痕相關(guān)基因的研究[J];中華創(chuàng)傷雜志;2001年06期

4 楊何義,應(yīng)萬濤,錢小紅;蛋白質(zhì)組技術(shù)的研究進(jìn)展[J];自然科學(xué)進(jìn)展;2002年01期

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本文編號：1686558