hLKB1基因重组质粒构建及蛋白表达和定位

目的构建hLKB1真核表达载体,证实融合蛋白在胃癌细胞内的表达及定位。方法提取胃正常黏膜上皮细胞GES-1的总mRNA并进行反转录。以反转录的cDNA为模板PCR扩增hLKB1全长编码基因,克隆至pCDNA3.1/flag的真核表达载体中。将构建的重组质粒测序并转染到胃癌SGC-7901细胞中,分别利用Western blot和激光共聚焦扫描显微技术检测其表达和在胃癌细胞中的定位。结果 hLKB1全长基因序列克隆到真核表达载体中,酶切鉴定片段为1300bp。Western blot检测到真核转染的Flag-hLKB1在胃癌SGC-7901细胞表达,条带约为50kD,免疫荧光显示蛋白广泛定位于细胞质和细胞核内。结论成功构建了hLKB1基因真核表达载体,并验证其在胃癌细胞表达。

Construction of fusion plasmid of hLKB1 gene and its recombinant protein expression and localization in gastric cancer

Objective To construct eukaryotic expression plasmid of hLKB 1 gene and identify its recombinant protein expression and localization in gastric cancer cells. Methods Total RNA was extracted from GES-1 cells, the hLKB1 coding sequence was amplified by polymerase chain reaction（PCR） method and cloned into pCDNA3.1/Flag vectors. After the target region was sequenced, the expression and localization of the pCDNA3.1/Flag-hLKB1 recombinant plasmid in SGC-7901 cells was proved by western blot and immunofluorescence. Results hLKB1 was cloned into pCDNA3.1/Flag successfully, the length of the fragment was about 1300bp. The Flag-hLKB1 expression in SGC-7901 cells was indentified by western blot and immunofluorescence with a molecular weight of 50kD. Conclusion The full length sequence of hLKB 1 was successfully cloned into eukaryotic expressing vectors and its fusion protein was expressed in SGC-7901 cells.