【摘要】：spindlin1是ssty/spin家族成員,編碼一個237個殘基的多肽鏈。它的功能和結(jié)構(gòu)基礎(chǔ)目前所知甚少。德國學(xué)者對已經(jīng)報告的spin/ssty家族成員進行了生物信息學(xué)分析,發(fā)現(xiàn)了一個Spin/Ssty repeat,該實驗組認(rèn)為這個Spin/Ssty repeat是一個新的motif、是獨立的功能單位,對該motif進一步分析,預(yù)測會形成一種未經(jīng)報道過的結(jié)構(gòu)---four-stranded beta-structure。 我們原核表達了GST融合的Spindlin1,親和純化、切去GST后經(jīng)陰離子交換色譜純化,得到了高純度的重組Spindlin1。我們采用氣相懸滴法獲得了可供X線衍射的晶體,經(jīng)過結(jié)晶條件優(yōu)化后,進行了同步輻射收集數(shù)據(jù)。衍射數(shù)據(jù)經(jīng)過軟件處理后我們得到了Spindlin1的空間結(jié)構(gòu),該結(jié)構(gòu)數(shù)據(jù)已經(jīng)提交到PDB數(shù)據(jù)庫,登錄號為1YDJ,目前數(shù)據(jù)還沒有釋放。 我們實驗組用真核表達載體pEGFP-N1-spindlin1轉(zhuǎn)染NIH 3T3細胞,對篩選出的穩(wěn)定轉(zhuǎn)染細胞進行了細胞生物學(xué)檢測。因此我們在生物信息學(xué)和晶體結(jié)構(gòu)的基礎(chǔ)上設(shè)計了突變體。真核突變載體已經(jīng)轉(zhuǎn)染NIH 3T3細胞,正在擴大培養(yǎng)。原核突變載體已經(jīng)交給清華大學(xué)結(jié)構(gòu)生物學(xué)實驗室。這部分工作將最終建立起結(jié)構(gòu)一功能的關(guān)系。
[Abstract]:Spindlin1 is a member of the ssty/spin family and encodes a polypeptide chain of 237residues. Little is known about its functional and structural basis. German scholars carried out bioinformatics analysis of reported members of the spin/ssty family and found a Spin/Ssty repeat,. The experimental group considered the Spin/Ssty repeat to be a new motif, as an independent functional unit, and further analyzed the motif. Predict that there will be an unreported structure-four-stranded beta-structure. We expressed GST fusion Spindlin1, affinity purification in prokaryotic. After GST was cut off, it was purified by anion exchange chromatography, and the recombinant Spindlin1. with high purity was obtained. The crystals which can be diffracted by X-ray diffraction were obtained by gas phase suspension drop method. After the crystallization conditions were optimized, the synchrotron radiation data were collected. After the diffraction data is processed by software, we get the spatial structure of Spindlin1, and the structure data has been submitted to PDB database, the login number is 1YDJ, and the data has not yet been released. In our experimental group, NIH 3T3 cells were transfected with eukaryotic expression vector pEGFP-N1-spindlin1, and the selected stable transfected cells were detected by cell biology. Therefore, we designed mutants on the basis of bioinformatics and crystal structure. Eukaryotic mutant vector has been transformed into NIH 3T3 cells and is being cultured. The prokaryotic mutation vector has been handed over to the Laboratory of structural Biology of Tsinghua University. This part of the work will eventually establish the relationship between structure and function.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R341

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