为了探索适用于体外培养的鱼细胞外源基因转入方法,本研究通过构建红鳍东方鲀(Takifugu rubripes)转录因子Sox2的重组表达载体 pET32a(+)-Sox2-11R-6His,诱导表达并纯化得到了 C末端连接多聚精氨酸(11R)的重组蛋白Sox2-11R-6His,以其与红鳍东方鲀精巢细胞系细胞共孵育12 h后,光镜观察结合Western Blot检测发现重组蛋白进入细胞的效率与浓度呈剂量依赖关系且最佳孵育浓度为8μg/mL,当重组蛋白质量浓度达到10μg/mL 时,表现出明显的细胞毒性。对外源蛋白进行免疫荧光标记定位,发现重组蛋白分布于细胞质,部分进入到细胞核中。证明了穿膜肽11R可以有效运载转录因子重组蛋白至红鳍东方鲀的细胞系细胞中。本研究旨在将广泛应用于哺乳动物的细胞基因递送载体穿膜肽应用于鱼类细胞系细胞。%Research on exogenous gene function in cultured cell line cells is guaranteed by two biological processes, efficient gene transduction and errorless gene expression. Fish cells are not easy for exogenous gene transduction and expression depending on the gene delivery method used and the transgene promoter. Cell penetrating peptides (CPPs), such as the CPP of the human immunodeficiency virus type 1 TAT protein, poly-arginine (6–12 residues), and the CPP derived from flock house virus (FHV), are short cationic and/or amphipathic peptides that are able to transport various biological compounds such as peptides, proteins, and oligonucleotides into mammalian cells to modulate biological activities inside cells. Although it has been shown that CPPs can mediate efficient delivery into a wide variety of mam-malian cell types, the transduction of proteins to cultured fish cells is considered more challenging. In this work, the Sox2 gene from Takifugu rupies genomic DNA was cloned and subsequent expression was carried out in Escherichia coli in the form of recombinant proteins by introducing cell penetrating peptides such as Sox2-11R-6His. The recombi-nant proteins were incubated with cultured T. rupies spermary cell line cells in vitro. Cells treated with proteins below 8μg/mL induced no or minor toxic effects while higher concentrations gave rise to cytotoxicity. The transduction effi-ciency of the recombinant protein with 6 His tag, evaluated by western blotting using an anti-His antibody, reached a maximum level at 8 μg/mL and in a dose responsive fashion. Based upon the optimal concentration of proteins for transduction, Sox2-11R-H6 in TRS cells was further imaged using a fluorescence microscope. The recombinant protein Sox2-11R-H6was detected in the cytoplasm and even in the nucleus. Taken together, the value of the 11R translocation domain in facilitating the routing of proteins to the cytosol of cultured fish cells was confirmed. However, whether the functionality of the transcription factor Sox2 was disturbed by addition of this domain in the construct needs further exploration. The subcellular localization of transduced proteins may depend on the nature of imported proteins, the cell type, and delivery approach. As a transcription factor, Sox2 must contain a nuclear localization signal (NLS) so that it can be transported to the nucleus to bind to its target octamer motif and transactivate its target genes. Our observation that fluorescence intensity was higher in the nucleus than cytoplasm, indicates that the exogenous protein Sox2 enters the fish nucleus. Further experiments are needed to decide whether nuclear location ofSox2-11R-H6 was facilitated by a nuclear localization signal or the cell penetrating peptide.
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