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巴马小型猪miR-183慢病毒载体的构建与鉴定

Construction and Identification of Lentiviral Vector Carrying Bama Mini-pig miR-183

  • 摘要: 本研究的目的在于构建猪pLV-miR-183慢病毒表达载体并检测其在猪肾细胞(PK15)中的表达情况。以巴马小型猪基因组DNA为模板,利用PCR克隆miR-183前体及部分侧翼序列,并使用T4连接酶将其连入p LV-CMV-MCS-EF1载体,构建pLV-miR-183慢病毒表达载体。将PCR和测序鉴定为阳性的miR-183重组质粒转染PK15细胞,利用荧光倒置显微镜检测其转染效率。结果显示,miR-183扩增序列长度为329 bp,与参考序列一致。将阳性的pLV-miR-183重组质粒转染PK15细胞48 h后,在荧光显微镜下检测到较强的绿色荧光蛋白表达,并通过qRT-PCR验证其较高的表达水平,说明所构建的pLV-miR-183慢病毒载体在PK15细胞中具有较高的表达活性。本研究成功构建了猪miR-183慢病毒表达载体,为后续研究miR-183在疾病作用机制方面奠定了一定的理论基础。

     

    Abstract: The aim of this study was to construct a porcine pLV-miR-183 lentiviral vector and detect its expression in porcine kidney cells(PK15). The genomic DNA of Guangxi Bama mini-pig was used as the template to clone the precursor and part of flanking sequences of miR-183 using PCR, and the sequence was inserted into pLV-CMV-MCS-EF1 vector by T4 ligase to construct the pLV-miR-183 lentiviral expression vector. The positive recombinant plasmids of miR-183 identified by PCR and sequencing were then transfected into PK15 cells, and the transfection efficiency was measured by fluorescence inverted microscope. The results showed that the length of miR-183 amplification sequence was 329 bp, which was consistent with the reference sequence. After transfection of positive recombinant plasmid pLV-miR-183 into PK15 cells for 48 h, strong expression of green fluorescent protein was detected under fluorescence microscopy, and its high expression level was verified by qRT-PCR,indicating that the constructed pLV-miR-183 lentiviral vector had high expression activity in PK15 cells. This study successfully constructed the porcine miR-183 lentiviral expression vector, which laid a certain theoretical foundation for the further study on the mechanism of disease action of miR-183.

     

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