基于Procaspase-8 DEDs的原核表达体系研究和肿瘤药物筛选细胞系研究

发布时间：2018-06-05 13:43

本文选题：半胱氨酸蛋白酶-8 + 定点突变　；参考：《昆明理工大学》2017年硕士论文

【摘要】：TRAIL(Tumor necrosis factor related apoptosis inducing ligand,TRAIL)作为肿瘤坏死因子家族的一员,因为其能选择性诱导肿瘤细胞、病毒感染细胞等发生凋亡,而对正常细胞没有明显的毒副作用,所以一直以来都受到研究者的关注。而且,随着研究的深入,越来越多的文献报道TRAIL除了抗肿瘤活性,还与阿尔茨海默病以及动脉粥样硬化等的发生发展有关。所以我们根据TRAIL凋亡通路中凋亡蛋白间的相互作用,希望获得凋亡蛋白解酶原-8死亡效应域蛋白(Procaspase-8 death effector domain,Procaspase-8 DEDs)与接头蛋白 FADD(Fas associated death domain)相互作用的关键氨基酸位点,这样就可以进行相应的拮抗药物设计,从而有助于阿尔茨海默病的治疗。同时,如果以Procaspase-8 DEDs为基础,构建用绿色荧光蛋白标记的Procaspase-8 DEDs真核细胞系,就可以用于筛选天然产物中具有抗肿瘤活性的药物分子。通过前期的实验研究发现无论是在真核细胞中还是原核细胞中,表达出来的野生型Procaspase-8 DEDs蛋白都不利于后续实验的顺利进行。后来通过大量的文献调研发现,因为Procaspase-8 DEDs本身的性质原因所以其会发生相互间的聚集,从而形成死亡效应纤丝(Death Effector Filaments,DEFs)。在真核细胞中,表达出来的野生型Procaspase-8 DEDs会自发的形成荧光聚集点,进而促使细胞凋亡,而在原核系统中对野生型的procaspase-8 DEDs质粒菌株进行诱导表达时.会因其易于聚集和低溶解性很难获得用于后续实验的可溶性目标蛋白单体。本实验室采用定点突变的方法对野生型(WT)Procaspase-8 DEDs蛋白编码基因的三个位点F122、L123、1128进行突变,并将获得的突变体基因片段构建入表达载体pET-28a(+)和pCMV6-AC-GFP中;在原核细胞中转化入E.Coli Transetta(DE3),获得具有三突变(F122G/L123G/I128D)的表达菌株,当以IPTG诱导目的基因表达时,发现突变蛋白(Procaspase-8 DEDs~(F122G/LI23G/I128D))的可溶性表达显著提高;当真核质粒转染细胞HCT116时,与野生型Procaspase-8 DEDs蛋白相比,突变型蛋白不利于死亡效应纤丝的形成。在预实验中,对转染了procaspase-8DEDsFI22G/LI23G/I128D真核荧光质粒的HepG2细胞株进行药物筛选测试,发现随着时间的变化有细胞会出现明显的绿色荧光聚集点。但是,后来发现同样处理的HepG2细胞不加TRAIL刺激时也会发生绿色荧光聚集点,而且细胞会死亡。综上所述,定点突变能提高Procaspase-8 DEDsF122G/L123G/I128D蛋白在大肠杆菌中的可溶性表达,并经Ni2+蛋白纯化柱和蛋白浓缩柱作用可富集可溶性目的蛋白;同时,在真核细胞中尽管一定程度上改善了死亡效应纤丝形成问题,但转染了procaspase-8 DEDs~(FI22GLI23GI128D)真核荧光质粒的细胞株若要用于药物筛选仍需要进一步优化实验方案。所以本文研究为基于Procaspase-8 DEDs蛋白的后续实验研究奠定了一定的基础。
[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.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

【参考文献】