人VMAT_2基因RNA探针制备及其在COS-7细胞中的表达

RNA PROBE PREPARATION OF CHINESE VESICULAR MONOAMINE TRANSPORTER-2 GENE AND ITS EXPRESSION IN COS-7 CELLS

本研究目的在于制备人 型囊泡单胺转运体 (VMAT2 )基因的反义和正义 RNA探针 ,以检测 VMAT2 基因能否在真核细胞表达。从携带 p GEM-Easy-T-VMAT2 克隆载体中通过限制性酶切反应得到 VMAT2 基因 ,与 p BK-RSV载体重组构建真核表达载体 p BK-RSV-VMAT2 ;分别以 T7和 T3聚合酶制备反义和正义探针 ,通过斑点杂交检测探针浓度。转染猴肾成纤维细胞COS-7,原位杂交及免疫荧光细胞化学检测 VMAT2 的表达。结果证明 ,反义和正义探针浓度分别为 80 ng/μl及 12 0 ng/μl;原位杂交及免疫组织化学证实重组的真核表达载体 p BK-RSV-VMAT2 在 COS-7的表达阳性率为 (10 .6± 1.2 ) %。本研究结果表明获得 VMAT2 基因反义链及正义链 RNA探针 ,并检测到 VMAT2

To prepare the RNA probe of Chinese human vesicular monoamine transporter 2(VMAT 2) gene and to detect whether the gene can express in eukaryotic cells, VMAT 2 gene derived from the clone vector pGEM Easy T VMAT 2 was subcloned into vector pBK RSV to construct the expression vector pBK RSV VMAT 2. We used pBK RSV VMAT 2 as a template to synthesize the RNA probe labelled digoxigenin with a transcriptional procedure in vitro, and determined the contents of probes by dot blot. After the recombinant vector was transiently transfected to COS 7 cells, the expression of VMAT 2 gene was detected by in situ hybridization and immunocytochemistry. The contents of antisense and sense probe were 80 ng/μl and 120 ng/μl respectively, and the ratio of positive cells of the gene in transfected COS 7 cells was (10.6±1.2)%. The results showed that the sensitive VMAT 2 RNA probes were obtained, and the Chinese VMAT 2 gene can be expressed in eukaryotic cells.