本文選題：半胱氨酸蛋白酶-8 + 定點突變�。� 參考：《昆明理工大學》2017年碩士論文

【摘要】：TRAIL(Tumor necrosis factor related apoptosis inducing ligand,TRAIL)作為腫瘤壞死因子家族的一員,因為其能選擇性誘導腫瘤細胞、病毒感染細胞等發(fā)生凋亡,而對正常細胞沒有明顯的毒副作用,所以一直以來都受到研究者的關注。而且,隨著研究的深入,越來越多的文獻報道TRAIL除了抗腫瘤活性,還與阿爾茨海默病以及動脈粥樣硬化等的發(fā)生發(fā)展有關。所以我們根據(jù)TRAIL凋亡通路中凋亡蛋白間的相互作用,希望獲得凋亡蛋白解酶原-8死亡效應域蛋白(Procaspase-8 death effector domain,Procaspase-8 DEDs)與接頭蛋白 FADD(Fas associated death domain)相互作用的關鍵氨基酸位點,這樣就可以進行相應的拮抗藥物設計,從而有助于阿爾茨海默病的治療。同時,如果以Procaspase-8 DEDs為基礎,構建用綠色熒光蛋白標記的Procaspase-8 DEDs真核細胞系,就可以用于篩選天然產物中具有抗腫瘤活性的藥物分子。通過前期的實驗研究發(fā)現(xiàn)無論是在真核細胞中還是原核細胞中,表達出來的野生型Procaspase-8 DEDs蛋白都不利于后續(xù)實驗的順利進行。后來通過大量的文獻調研發(fā)現(xiàn),因為Procaspase-8 DEDs本身的性質原因所以其會發(fā)生相互間的聚集,從而形成死亡效應纖絲(Death Effector Filaments,DEFs)。在真核細胞中,表達出來的野生型Procaspase-8 DEDs會自發(fā)的形成熒光聚集點,進而促使細胞凋亡,而在原核系統(tǒng)中對野生型的procaspase-8 DEDs質粒菌株進行誘導表達時.會因其易于聚集和低溶解性很難獲得用于后續(xù)實驗的可溶性目標蛋白單體。本實驗室采用定點突變的方法對野生型(WT)Procaspase-8 DEDs蛋白編碼基因的三個位點F122、L123、1128進行突變,并將獲得的突變體基因片段構建入表達載體pET-28a(+)和pCMV6-AC-GFP中;在原核細胞中轉化入E.Coli Transetta(DE3),獲得具有三突變(F122G/L123G/I128D)的表達菌株,當以IPTG誘導目的基因表達時,發(fā)現(xiàn)突變蛋白(Procaspase-8 DEDs~(F122G/LI23G/I128D))的可溶性表達顯著提高;當真核質粒轉染細胞HCT116時,與野生型Procaspase-8 DEDs蛋白相比,突變型蛋白不利于死亡效應纖絲的形成。在預實驗中,對轉染了procaspase-8DEDsFI22G/LI23G/I128D真核熒光質粒的HepG2細胞株進行藥物篩選測試,發(fā)現(xiàn)隨著時間的變化有細胞會出現(xiàn)明顯的綠色熒光聚集點。但是,后來發(fā)現(xiàn)同樣處理的HepG2細胞不加TRAIL刺激時也會發(fā)生綠色熒光聚集點,而且細胞會死亡。綜上所述,定點突變能提高Procaspase-8 DEDsF122G/L123G/I128D蛋白在大腸桿菌中的可溶性表達,并經Ni2+蛋白純化柱和蛋白濃縮柱作用可富集可溶性目的蛋白;同時,在真核細胞中盡管一定程度上改善了死亡效應纖絲形成問題,但轉染了procaspase-8 DEDs~(FI22GLI23GI128D)真核熒光質粒的細胞株若要用于藥物篩選仍需要進一步優(yōu)化實驗方案。所以本文研究為基于Procaspase-8 DEDs蛋白的后續(xù)實驗研究奠定了一定的基礎。
[Abstract]:As a member of the tumor necrosis factor family, TRAIL(Tumor necrosis factor related apoptosis inducing ligand trail can selectively induce apoptosis in tumor cells and virus infected cells, but has no obvious toxicity to normal cells. So it has been paid attention by researchers all the time. Moreover, with the development of research, more and more literatures have reported that TRAIL is related to the occurrence and development of Alzheimer's disease and atherosclerosis in addition to its antitumor activity. Therefore, according to the interaction between apoptotic proteins in TRAIL apoptosis pathway, we hope to obtain the key amino acid site of the interaction between Procaspase-8 death effector domain protein Procaspase-8 DEDsand FADD(Fas associated death domain). This allows for the design of appropriate antagonistic drugs, thus contributing to the treatment of Alzheimer's disease. At the same time, if the Procaspase-8 DEDs eukaryotic cell line labeled with green fluorescent protein is constructed on the basis of Procaspase-8 DEDs, it can be used to screen anti-tumor drug molecules in natural products. It was found that the wild-type Procaspase-8 DEDs protein expressed in eukaryotic cells and prokaryotic cells was unfavorable for further experiments. After a lot of literature research, it was found that Procaspase-8 DEDs would gather together because of its own nature, thus forming death Effector filamentsof DEFs. In eukaryotic cells, the expressed wild-type Procaspase-8 DEDs will spontaneously form fluorescent aggregates, and then promote cell apoptosis. In the prokaryotic system, the wild-type procaspase-8 DEDs plasmid strain is induced to express. It is difficult to obtain soluble target protein monomer for further experiments because of its easy aggregation and low solubility. In our laboratory, we used site-directed mutagenesis to mutate the three loci of the encoding gene of the wild type WTT Procaspase-8 DEDs protein, and constructed the obtained mutant gene fragments into the expression vectors pET-28a () and pCMV6-AC-GFP. When the target gene expression was induced by IPTG, it was found that the soluble expression of the mutant protein Procaspase-8 DEDX F122G / I122GL23 / I128D was significantly increased, and when the eukaryotic plasmid was transfected into the cell HCT116, the expression of the mutant protein Procaspase-8 DEDX / F122GL23 / I128D was significantly increased when IPTG was used to induce the expression of the target gene. Compared with wild-type Procaspase-8 DEDs protein, mutant protein is not conducive to the formation of death effect filaments. In the pre-experiment, the HepG2 cell lines transfected with procaspase-8DEDsFI22G/LI23G/I128D eukaryotic fluorescence plasmid were screened for drugs, and it was found that there were obvious green fluorescent aggregates in the cells with the change of time. However, it was later found that HepG2 cells treated with the same treatment also had green fluorescent aggregates without TRAIL stimulation, and the cells died. In conclusion, site-directed mutation can improve the soluble expression of Procaspase-8 DEDsF122G/L123G/I128D protein in Escherichia coli, and can enrich soluble target protein by Ni2 protein purification column and protein concentration column. In eukaryotic cells, although the death effect filamentous formation was improved to a certain extent, the cell lines transfected with procaspase-8 DEDsSU FI22GLI23GI128D) still need to be further optimized for drug screening. Therefore, this study has laid a foundation for further experimental research based on Procaspase-8 DEDs protein.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R91

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