红鳍东方鲀重编程因子Oct4和Sox2原核表达载体的构建、精巢细胞原核表达产物的导入及其检测研究
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摘要
重编程因子Oct4与Sox2作为转录因子,可通过开启相关基因的转录诱导体细胞的重编程,在诱导型多能干细胞(iPS细胞)的制备及相关研究中具有重要的应用价值。目前,获得哺乳动物iPS细胞的最安全的途径是直接导入外源重编程因子的编码蛋白。为了获得红鳍东方鲀精巢细胞的iPS细胞,为优质、高产、抗逆红鳍东方鲀的细胞工程育种奠定基础,本文利用pET32a与穿模肽构建出Oct4和Sox2的原核表达载体,通过转化大肠杆菌(E. coli) BL21诱导表达出其编码蛋白,经与体外培养的红鳍东方鲀精巢细胞系细胞共孵育将Oct4和Sox2的编码蛋白导入精巢细胞,并对导入编码蛋白的细胞内定位进行了检测。
根据红鳍东方鲀基因组数据库中的已公布序列设计Oct4和Sox2引物,从红鳍东方鲀肝组织中克隆出Oct4和Sox2基因,在其两端分别引入BamH I和Kpn I限制性酶切位点,在合成的由11个精氨酸组成的细胞穿膜肽序列两端引入Kpn I和EcoR I限制性酶切位点,利用Kpn I酶切位点的粘性末端将穿膜肽序列分别连接到Oct4和Sox2基因的3'端上,进而利用Oct4和Sox2基因的BamH I酶切位点以及穿膜肽序列的EcoR I酶切位点将Oct4基因、Sox2基因和穿膜肽序列连到pET32a原核表达载体中,构建出pET32a-Oct4-11R和pET32a-Sox2-11R表达质粒。将上述构建的表达载体分别转化到E. coli BL21中,分别在30℃利用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导处理5 h进行Oct4和Sox2基因编码产物的诱导表达,经过超声波和冻融破碎细胞后,对存在于细胞质中的Oct4重组蛋白表达产物直接采用Ni-NTA树脂进行纯化,对以包涵体形式存在的Sox2重组蛋白表达产物经尿素变性处理后再采用Ni-NTA树脂进行纯化,经透析获得复性的纯化蛋白,经过过滤除菌后,添加到红鳍东方鲀精巢细胞的培养液中,孵育精巢细胞过夜,光学显微镜下观察细胞形态及Western blot检测结果显示,Oct4和Sox2重组蛋白与细胞作用的最佳浓度均为8μg/ml。免疫荧光检测结果发现,导入的Oct4和Sox2重组蛋白在细胞质和细胞核中均有分布,但主要集中细胞核中,表明这两种重组蛋白均能在穿膜肽的引导下进入到细胞核中。上述实验结果证实,本文所构建的Oct4和Sox2基因表达载体能在E. coli中得到有效表达,且所引入的穿膜肽能有效介导Oct4和Sox2重组蛋白表达产物穿膜进入红鳍东方鲀精巢细胞核,导入后的红鳍东方鲀精巢细胞现已存活了10天,为红鳍东方鲀精巢细胞诱导转化为iPS细胞奠定了基础。
Oct4 and Sox2, as transcription factors, play an important role in inducing pluripotent stem cells (iPS cells) by activating related genes. At present, it has been demonstrated that mammal iPS cells can be obtained safely by directly delivering the proteins of reprogramming factors to somatic cells. This research is the preliminary stage of inducing Takifugu rubripes testicular cells to iPS cells, which will further lay foundation for cell engineering and breeding. Transcription factors Oct4 and Sox2 were connected with cell penetrating peptide sequence to get the recombinant transcription factor proteins, which were then transfered into in vitro cultured Takifugu rubripes testicular cells. The intracellular location of the recombinant protein was determined.
The primers of Oct4 and Sox2 were designed by the published genome database of Takifugu rubripes. The two genes, cloned from the liver of Takifugu rubripes, were introduced restriction enzyme sites BamH I and Kpn I at both ends. Similarly, the both ends of the synthesized cell penetrating peptide sequence (11R), composed of 11 arginines transduction domain, were introduced restriction enzyme sites Kpn I and EcoR I. The 11R were connected to the 3’terminal of reprogramming factors after both digestion by the restriction enzyme Kpn I. The product was then ligased to the prokaryotic expression vector pET32a after both digestion by BamH I and EcoR I to construct the expression plasmids pET32a-oct4-11R and pET32a-sox2-11R. The two recombinant plasmids were transformed into E.coli BL21(DE3) and the expression were induced by Isopropylβ-D-1-Thiogalactopyranoside (IPTG) for 5h at 30℃. Then E.coli cells were broken by freeze-thaw and ultrasonic. The recombinant proteins Oct4, which present in the cytoplasm, were purified by Ni-NTA resin. The Sox2, in the form of inclusion bodies, were denatured by urea firstly, purified using Ni-NTA resin, and renaturation by dialysis. After sterilization by filtration, the two transcription factor proteins were added to the culture medium of Takifugu rubripes testicular cells. Through observation of cell morphology under light microscope and Western blot detection of recombinant proteins, the optimal concentrations of the recombinant proteins were determined at 8μg/ml. The proteins could be observed distributing throughout the cell by immunofluorescence labeling, especially focus on the nucleus. It indicates that the two recombinant proteins can enter the nucleus mediated by the cell penetrating peptide.
The above results confirmed that the constructed expression vectors of Oct4 and Sox2 can be effectively expressed in E. coli. The recombinant proteins of Oct4 and Sox2 were introduced effectively into the nucleus of Takifugu rubripes testicular cells mediated by the penetrating peptide. Cells have survived for 10 days now with recombinant proteins in them, which lay early foundation for further inducing Takifugu rubripes testicular cells to iPS cells.
根据红鳍东方鲀基因组数据库中的已公布序列设计Oct4和Sox2引物,从红鳍东方鲀肝组织中克隆出Oct4和Sox2基因,在其两端分别引入BamH I和Kpn I限制性酶切位点,在合成的由11个精氨酸组成的细胞穿膜肽序列两端引入Kpn I和EcoR I限制性酶切位点,利用Kpn I酶切位点的粘性末端将穿膜肽序列分别连接到Oct4和Sox2基因的3'端上,进而利用Oct4和Sox2基因的BamH I酶切位点以及穿膜肽序列的EcoR I酶切位点将Oct4基因、Sox2基因和穿膜肽序列连到pET32a原核表达载体中,构建出pET32a-Oct4-11R和pET32a-Sox2-11R表达质粒。将上述构建的表达载体分别转化到E. coli BL21中,分别在30℃利用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导处理5 h进行Oct4和Sox2基因编码产物的诱导表达,经过超声波和冻融破碎细胞后,对存在于细胞质中的Oct4重组蛋白表达产物直接采用Ni-NTA树脂进行纯化,对以包涵体形式存在的Sox2重组蛋白表达产物经尿素变性处理后再采用Ni-NTA树脂进行纯化,经透析获得复性的纯化蛋白,经过过滤除菌后,添加到红鳍东方鲀精巢细胞的培养液中,孵育精巢细胞过夜,光学显微镜下观察细胞形态及Western blot检测结果显示,Oct4和Sox2重组蛋白与细胞作用的最佳浓度均为8μg/ml。免疫荧光检测结果发现,导入的Oct4和Sox2重组蛋白在细胞质和细胞核中均有分布,但主要集中细胞核中,表明这两种重组蛋白均能在穿膜肽的引导下进入到细胞核中。上述实验结果证实,本文所构建的Oct4和Sox2基因表达载体能在E. coli中得到有效表达,且所引入的穿膜肽能有效介导Oct4和Sox2重组蛋白表达产物穿膜进入红鳍东方鲀精巢细胞核,导入后的红鳍东方鲀精巢细胞现已存活了10天,为红鳍东方鲀精巢细胞诱导转化为iPS细胞奠定了基础。
Oct4 and Sox2, as transcription factors, play an important role in inducing pluripotent stem cells (iPS cells) by activating related genes. At present, it has been demonstrated that mammal iPS cells can be obtained safely by directly delivering the proteins of reprogramming factors to somatic cells. This research is the preliminary stage of inducing Takifugu rubripes testicular cells to iPS cells, which will further lay foundation for cell engineering and breeding. Transcription factors Oct4 and Sox2 were connected with cell penetrating peptide sequence to get the recombinant transcription factor proteins, which were then transfered into in vitro cultured Takifugu rubripes testicular cells. The intracellular location of the recombinant protein was determined.
The primers of Oct4 and Sox2 were designed by the published genome database of Takifugu rubripes. The two genes, cloned from the liver of Takifugu rubripes, were introduced restriction enzyme sites BamH I and Kpn I at both ends. Similarly, the both ends of the synthesized cell penetrating peptide sequence (11R), composed of 11 arginines transduction domain, were introduced restriction enzyme sites Kpn I and EcoR I. The 11R were connected to the 3’terminal of reprogramming factors after both digestion by the restriction enzyme Kpn I. The product was then ligased to the prokaryotic expression vector pET32a after both digestion by BamH I and EcoR I to construct the expression plasmids pET32a-oct4-11R and pET32a-sox2-11R. The two recombinant plasmids were transformed into E.coli BL21(DE3) and the expression were induced by Isopropylβ-D-1-Thiogalactopyranoside (IPTG) for 5h at 30℃. Then E.coli cells were broken by freeze-thaw and ultrasonic. The recombinant proteins Oct4, which present in the cytoplasm, were purified by Ni-NTA resin. The Sox2, in the form of inclusion bodies, were denatured by urea firstly, purified using Ni-NTA resin, and renaturation by dialysis. After sterilization by filtration, the two transcription factor proteins were added to the culture medium of Takifugu rubripes testicular cells. Through observation of cell morphology under light microscope and Western blot detection of recombinant proteins, the optimal concentrations of the recombinant proteins were determined at 8μg/ml. The proteins could be observed distributing throughout the cell by immunofluorescence labeling, especially focus on the nucleus. It indicates that the two recombinant proteins can enter the nucleus mediated by the cell penetrating peptide.
The above results confirmed that the constructed expression vectors of Oct4 and Sox2 can be effectively expressed in E. coli. The recombinant proteins of Oct4 and Sox2 were introduced effectively into the nucleus of Takifugu rubripes testicular cells mediated by the penetrating peptide. Cells have survived for 10 days now with recombinant proteins in them, which lay early foundation for further inducing Takifugu rubripes testicular cells to iPS cells.
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