發(fā)布時(shí)間：2018-06-05 13:43

  本文選題：半胱氨酸蛋白酶-8 + 定點(diǎn)突變�。� 參考：《昆明理工大學(xué)》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?yàn)槠淠苓x擇性誘導(dǎo)腫瘤細(xì)胞、病毒感染細(xì)胞等發(fā)生凋亡,而對(duì)正常細(xì)胞沒(méi)有明顯的毒副作用,所以一直以來(lái)都受到研究者的關(guān)注。而且,隨著研究的深入,越來(lái)越多的文獻(xiàn)報(bào)道TRAIL除了抗腫瘤活性,還與阿爾茨海默病以及動(dòng)脈粥樣硬化等的發(fā)生發(fā)展有關(guān)。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(yīng)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(guān)鍵氨基酸位點(diǎn),這樣就可以進(jìn)行相應(yīng)的拮抗藥物設(shè)計(jì),從而有助于阿爾茨海默病的治療。同時(shí),如果以Procaspase-8 DEDs為基礎(chǔ),構(gòu)建用綠色熒光蛋白標(biāo)記的Procaspase-8 DEDs真核細(xì)胞系,就可以用于篩選天然產(chǎn)物中具有抗腫瘤活性的藥物分子。通過(guò)前期的實(shí)驗(yàn)研究發(fā)現(xiàn)無(wú)論是在真核細(xì)胞中還是原核細(xì)胞中,表達(dá)出來(lái)的野生型Procaspase-8 DEDs蛋白都不利于后續(xù)實(shí)驗(yàn)的順利進(jìn)行。后來(lái)通過(guò)大量的文獻(xiàn)調(diào)研發(fā)現(xiàn),因?yàn)镻rocaspase-8 DEDs本身的性質(zhì)原因所以其會(huì)發(fā)生相互間的聚集,從而形成死亡效應(yīng)纖絲(Death Effector Filaments,DEFs)。在真核細(xì)胞中,表達(dá)出來(lái)的野生型Procaspase-8 DEDs會(huì)自發(fā)的形成熒光聚集點(diǎn),進(jìn)而促使細(xì)胞凋亡,而在原核系統(tǒng)中對(duì)野生型的procaspase-8 DEDs質(zhì)粒菌株進(jìn)行誘導(dǎo)表達(dá)時(shí).會(huì)因其易于聚集和低溶解性很難獲得用于后續(xù)實(shí)驗(yàn)的可溶性目標(biāo)蛋白單體。本實(shí)驗(yàn)室采用定點(diǎn)突變的方法對(duì)野生型(WT)Procaspase-8 DEDs蛋白編碼基因的三個(gè)位點(diǎn)F122、L123、1128進(jìn)行突變,并將獲得的突變體基因片段構(gòu)建入表達(dá)載體pET-28a(+)和pCMV6-AC-GFP中;在原核細(xì)胞中轉(zhuǎn)化入E.Coli Transetta(DE3),獲得具有三突變(F122G/L123G/I128D)的表達(dá)菌株,當(dāng)以IPTG誘導(dǎo)目的基因表達(dá)時(shí),發(fā)現(xiàn)突變蛋白(Procaspase-8 DEDs~(F122G/LI23G/I128D))的可溶性表達(dá)顯著提高;當(dāng)真核質(zhì)粒轉(zhuǎn)染細(xì)胞HCT116時(shí),與野生型Procaspase-8 DEDs蛋白相比,突變型蛋白不利于死亡效應(yīng)纖絲的形成。在預(yù)實(shí)驗(yàn)中,對(duì)轉(zhuǎn)染了procaspase-8DEDsFI22G/LI23G/I128D真核熒光質(zhì)粒的HepG2細(xì)胞株進(jìn)行藥物篩選測(cè)試,發(fā)現(xiàn)隨著時(shí)間的變化有細(xì)胞會(huì)出現(xiàn)明顯的綠色熒光聚集點(diǎn)。但是,后來(lái)發(fā)現(xiàn)同樣處理的HepG2細(xì)胞不加TRAIL刺激時(shí)也會(huì)發(fā)生綠色熒光聚集點(diǎn),而且細(xì)胞會(huì)死亡。綜上所述,定點(diǎn)突變能提高Procaspase-8 DEDsF122G/L123G/I128D蛋白在大腸桿菌中的可溶性表達(dá),并經(jīng)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(shí),在真核細(xì)胞中盡管一定程度上改善了死亡效應(yīng)纖絲形成問(wèn)題,但轉(zhuǎn)染了procaspase-8 DEDs~(FI22GLI23GI128D)真核熒光質(zhì)粒的細(xì)胞株若要用于藥物篩選仍需要進(jìn)一步優(yōu)化實(shí)驗(yàn)方案。所以本文研究為基于Procaspase-8 DEDs蛋白的后續(xù)實(shí)驗(yàn)研究奠定了一定的基礎(chǔ)。
[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(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R91

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