猪LIN28、KLF4、c-MYC和OCT4基因原核表达载体构建及重组蛋白制备

发布时间：2018-03-20 22:13

本文选题：猪　切入点：多能性转录因子　出处：《安徽农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：诱导多能干细胞(Induced Pluripotent Stem Cells,iPSCs)自诞生以来,由于和胚胎干细胞(Embryonic Stem Cells,ESCs)一样既有自我更新、无限增殖能力,又有分化形成机体几乎全部类型细胞的潜能,在再生医学、畜禽种质资源保护与创新和生物学基础研究等方面极具应用价值,因而迅速成为生命科学研究领域中的前沿热点。如今,人们已成功获得了包括猪在内的多种家养动物的iPSCs,打破了长期未能获取猪和其它家畜真正ESCs的窘境。然而,iPSCs仍面临质量参差不齐、效率低和安全性不确定等突出问题。利用重组蛋白或小分子化合物诱导iPSCs为克服外源基因插入所可能导致的安全隐患奠定了良好基础。但至今关于重组蛋白诱导猪iPSCs的报道十分有限。本研究将分别构建猪LIN28、KLF4、c-MYC和OCT4四种基因与细胞穿膜肽11R融合表达的原核载体,并重点探讨制备猪LIN28-11R可溶性融合重组蛋白的优化条件,同时对另三种转录因子重组蛋白的表达条件进行初步探索,以期为今后利用这四种重组蛋白诱导获取猪iPSCs奠定基础。具体试验及结果如下:1.猪LIN28、KLF4、c-MYC及OCT4基因原核表达载体的构建及鉴定。首先,用Optimum Gene软件对猪OCT4(NP_001106531)、KLF4(NP_001026952)、c-MYC(NP_001005154)、LIN28(NP_001116605)基因序列进行密码子优化,并在目的基因3’端加上编码Linker和穿膜肽11R的基因序列。通过人工合成的方法获得目的基因,再亚克隆至pET30a载体中。酶切鉴定和测序验证结果表明,成功构建出pET30a-NdeI-Oct4-linker-11R-XhoI-His、pET30a-NdeI-Lin28-linker-11R-XhoI-His、pET30a-Nde I-Klf4-linker-11R-XhoI-His、pET30a-NdeI-c-Myc-linker-11R-Xho I-His四种穿膜肽与多能性转录因子融合表达的原核载体。2.猪源LIN28-11R重组蛋白制备及其它三种转录因子重组蛋白诱导表达条件的初步探索。将重构质粒转化到大肠杆菌BL21中,在不同的时间和温度条件下诱导表达,筛选出LIN28-11R重组蛋白的最佳表达条件,并进行大量表达;蛋白产物经纯化和鉴定后进行细胞穿膜添加实验,以验证其是否具有穿透细胞膜的能力。同时,对另外三种多能性因子重组蛋白的诱导表达条件进行了初步探索。本试验最终获得并纯化出了可溶性的猪LIN28-11R重组蛋白,该重组蛋白具有穿透细胞膜的能力。而初步研究发现,OCT4-11R、KLF4-11R和c-MYC-11R三种重组蛋白的最佳诱导表达条件可能是:KLF4-11R在37℃诱导4h时表达量相对较高,并在菌体裂解液上清中表达量较高;c-MYC-11R在37℃诱导4h的条件下有表达,且只在菌体裂解液及裂解液沉淀中表达;OCT4-11R在不同温度(37℃、30℃)条件下几乎均未表达。总之,本研究成功构建了猪LIN28、KLF4、c-MYC和OCT4多能性转录因子与细胞穿膜肽融合表达的原核载体,成功表达纯化出了猪LIN28-11R可溶性融合重组蛋白,且融合蛋白具有穿透细胞膜的能力。此外,初步确定了OCT4-11R、KLF4-11R和c-MYC-11R三种转录因子重组蛋白的诱导表达条件。本研究为其它重组蛋白的制备提供了依据,并为今后利用重组蛋白诱导猪iPSCs奠定了基础。
[Abstract]:Induced pluripotent stem cells (Induced Pluripotent Stem Cells, iPSCs) since its birth, and because embryonic stem cells (Embryonic Stem Cells, ESCs) as both self-renewal, unlimited proliferation, differentiation and formation of the body almost all cell types in the potential for regenerative medicine, animal protection and innovation of germplasm resources and biology the basic research of high value, which quickly became the hotspot in the field of life science. Nowadays, people have been successfully obtained including pigs including a variety of domestic animal iPSCs, breaking the long failed to get real ESCs the dilemma of pigs and other livestock. However, iPSCs still faces uneven quality, low efficiency and safety determination of the outstanding problems. Induction of iPSCs laid a good foundation for security risks could lead to overcome foreign gene insertion using recombinant protein or small molecule compounds. So far about pigs induced by iPSCs recombinant protein reported is very limited. This study will construct porcine LIN28, KLF4, prokaryotic vector c-MYC and OCT4 four kinds of gene and cell penetrating peptide 11R fusion expression, and focus on the optimization conditions of preparation of porcine LIN28-11R soluble fusion recombinant protein expression, while the other three conditions of transcription the recombinant protein factor are explored, with a view to the future use of the four kinds of recombinant porcine iPSCs. Get laid the foundation concrete trials and results are as follows: 1. porcine LIN28, KLF4, and the carrier phase to construct the prokaryotic expression of c-MYC gene and OCT4 gene of porcine OCT4. Firstly, using Optimum Gene software (NP_001106531). KLF4 (NP_001026952), c-MYC (NP_001005154), LIN28 (NP_001116605) gene sequence of codon optimization, and in the 3 'end with gene encoding Linker and transmembrane peptide 11R gene sequences obtained by synthetic methods. The target gene, and then subcloned into pET30a vector. Enzyme digestion and sequencing results showed that successfully constructed pET30a-NdeI-Oct4-linker-11R-XhoI-His, pET30a-NdeI-Lin28-linker-11R-XhoI-His, pET30a-Nde I-Klf4-linker-11R-XhoI-His, pET30a-NdeI-c-Myc-linker-11R-Xho I-His four cell penetrating peptides and can explore the expression conditions of induced transcription factor fusion protein prokaryotic vector.2. of porcine LIN28-11R and other recombinant protein preparation three kinds of transcription factors of the recombinant protein. The reconstructed plasmid transformed into E.coli BL21 and induced expression in time and different temperature conditions, screening of recombinant protein LIN28-11R optimal expression conditions, and a number of expression; protein after purification and identification of cell penetrating addition experiments to verify its ability penetrate the cell membrane. At the same time, for the other three pluripotency by recombinant Explores the expression conditions of protein. This experiment obtained and purified soluble recombinant protein of porcine LIN28-11R, the recombinant protein has the ability to penetrate cell membranes and OCT4-11R. The preliminary study found that, three kinds of recombinant protein KLF4-11R and c-MYC-11R induce the expression of the best conditions may be relatively high expression of KLF4-11R in 37 C induced by 4h, and high expression in cell lysate supernatant; expression of c-MYC-11R at 37 DEG 4H induced conditions, and only expressed in cell lysate and lysate precipitation; OCT4-11R at different temperature (37 DEG, 30 DEG C) condition are almost no expression. In conclusion, this study the successful construction of porcine LIN28, KLF4, c-MYC and OCT4 can be prokaryotic transcription factors and cell penetrating peptide fusion protein, successfully expressed and purified porcine LIN28-11R soluble fusion recombinant protein and fusion protein with penetration The ability of cell membrane was also identified. In addition, the induced expression conditions of three transcription factors, OCT4-11R, KLF4-11R and c-MYC-11R, were preliminarily determined. This study provides a basis for the preparation of other recombinant proteins, and lays the foundation for the future use of recombinant protein to induce pig iPSCs.

【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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