大鼠辣椒素受体的重组

目的 构建辣椒素受体（vanilloid receptor1，VR1）真核表达载体，用以研究VR1的功能。方法 从大鼠的背根神经节中提取总RNA，逆转录为cDNA，以此为模板，用VR1特异性的引物扩增，随后将PCR产物插入T-easy载体，经酶切鉴定和测序，与GenBank序列比对。再将正确序列亚克隆至表达载体pEGFP-C3并鉴定。运用脂质体转染的方法，将构建好的表达载体pEGFP-C3-VR1转染到人胚胎肾细胞（HEK-293）中，用荧光显微镜观察报告基因绿色荧光蛋白的表达，通过免疫组化的方法确定目的蛋白VR1的表达。结果 克隆的大鼠VR1核苷酸序列与GenBank公布的序列AB040873和AF029310的编码区同源性分别达到99.92%和99.84%。转染24h后，绿色荧光蛋白和VR1均表达。结论 重组的VR1真核表达载体构建基本正确，并在HEK-293细胞中成功表达，为进一步研究其功能作了准备。

Recombination of vanilloid receptor 1 in rats

Objective To construct the expression vector of vanilloid receptor 1 in rats, and to explore its function. Methods The cDNA of VR1, from the dorsal mot ganglion of rat, was amplified by PCR, cloned into T-easy vector, and then identified and sequenced. The correct clone was subcloned into pEGFP-C3 vector, and amplified in E. coli JM109. Then the recombinant pEGFP- C3-VR1 vectors were harvested and identified by sequencing. The plasmid pEGFP-C3-VR1 was transfected to HEK-293, a human embryonic kidney cell, by liposome reagent. The positive cells were identified by detecting the report gene, green fluorescence protein, and by immunohistochemistry assay. Results DNA sequence analysis showed that the nucleic acid sequence homological rates of cloned rat VR1 to the code region of AB040873 and AF029311 published in GeneJ3ank were 99.92% and 99.84%, respectively.Green fluorescence protein and VR1 also expressed in HEK-293 after transfection for 24 h. Conclusion The recombinant plasmid of rat VR1 is successfully constructed and expressed in HEK-293 cells, which would make the further study of VR1 possible.