五羟色胺-N-乙酰转移酶基因真核表达载体的构建、表达及活性检测

目的构建pTARGET^TM—AANAT真核表达载体并转化L6成肌细胞，检测目的基因的表达并初步探讨AANAT转基因细胞的生物活性。方法提取大鼠松果体组织mRNA，RT-PCR技术扩增五羟色胺-N-乙酰转移酶基因（AANAT）cDNA，酶切连接pTARGET^TM载体，脂质体法导入L6成肌细胞。倒置显微镜观察转化细胞体外存活、RT-PCR及Western blotting技术检测pTARGET^TM-AANAT的基因表达产物及转化细胞的功能表达。结果酶切分析、DNA测序及凝胶电泳检测结果证实，pTARGET^TM-AANAT真核表达载体构建成功。RT-PCR及Western blotting检测表明，转化后的L6细胞表达AANAT mRNA，细胞具备AANAT蛋白的表达能力。结论成功构建pTARGET^TM-AANAT的真核表达载体，转化的L6细胞株能表达AANAT蛋白活性。

Construction, Expression and Detection of the Eukaryotic Expression Vector of Serotonin-N-Acetyltransferase Gene

Objective To investigate the possibility that the construction and expression of a eukaryotic expression vector system of rat NN-NAT gene. Methods The full-length eDNA fragment of rat AA-NAT gene was amplified by RT-PCR method. After retrieving the PCR products, ligating it with pTARGET^TM vector, transformating ligation reaction to JM109 huge efficiency competent cells and identifying the recombinant plasmid, the recombinant eukaryotie expression vector pTARGE^TM-AANAT was transfected into rat 1.6 myoblasts with lipofectamine. Accordingly, engineered cells selected by antibiotic G418 were detected by the methods of RT-PCR and Western blotting. Results It was revealed that, amplified AA-NAT eDNA confirmed by agarose gel eleetrophoresis could ligate with pTARGET^TM vector and subcloned into JM109 cells. 1.6 cells transfeeted with pTARGE^TM-AA- NAT survived well after G418 selection and expressed AA-NAT protein. Conclusion Our results suggest that we have prepared rat AA-NAT stable eukaryotie expression system successfully although it was just a primary result. This system can be used for the transfeetion of 1.6 myoblast.