正义和反义β1,4-半乳糖基转移酶V表达质粒在星形胶质细胞瘤细胞中的表达与鉴定

Construction and transfection of expression plasmids of β1,4 - galactosyltransferase V in SHG-44 cells

目的构建β1,4-半乳糖基转移酶V(β1,4-galactosyltransferase V,β-1,4-GalT-V)正、反义表达质粒,为探讨神经胶质瘤细胞表达β-1,4-GalT-V的生物学意义提供研究基础。方法构建β-1,4-GalT-V表达质粒,并将其转染到人脑星形胶质细胞瘤细胞SHG-44中;用Western blot和Northern blot鉴定转染后基因表达情况。结果通过酶切鉴定和测序分析证实,成功构建了β-1,4-GalT-V表达质粒。将构建的表达质粒转染到胶质细胞瘤细胞SHG-44中,正义表达质粒能增加胶质细胞瘤细胞SHG-44中的β-1,4-GalT-V表达量,而转染反义质粒则能降低细胞中β-1,4-GalT-V表达量。结论正、反义β-1,4-GalT-V表达质粒在人脑星形胶质细胞瘤细胞SHG-44中的稳定转染和表达,对分析胶质瘤细胞表达β-1,4-GalT-V的意义以及对肿瘤细胞的增殖、迁移和黏附等的影响,提供了研究基础。

Objective To study the biological effects of β1,4-galactosyltransferase V （β -1,4- GaIT-V） expressed in astrocytoma, the sense and antisense expression plasmids of β1, 4-GalT-V were constructed and transfected into SHG-44 cells, one kind of astrocytoma cell lines. Methods The whole gene sequence of β1, 4-GalT-V was cloned into the pGEM-T vector and subcloned into the expression plasmid pcDNA3. 1. In order to construct the antisense plasmid of β1,4-GaIT-V, the frag- ment from the start code ATG of β1,4-GalT-V was generated by PCR using the antisense primers. The plasmid containing sense or antisense β1,4-GalT-V was verified by EcoR I / Xho I digestion and DNA sequencing. The constructed expression plasmids were transfected into SHG-44 cells and certificated by Western blot and Northern blot analysis. Results It was found that the sense and antisense expression plasmids of β1,4-GalT-V were constructed and transfected into the SHG-44 cells success- fully. The plasmid containing sense β1,4-GalT-V could increase the expression of β1,4-GaIT-V, but the plasmid containing antisense β1, 4-GalT-V could decrease the expression of β1, 4-GalT-V. Conclusion The astrocytoma cells transfected with the expression plasmids of β1,4-GalT-V will help to study the effects of β1,4-GalT-V on proliferation, adhesion and migration of astrocytoma cells.