CONSTRUCTION,EXPRESSION AND DETECTION OF THE EUKARYOTIC EXPRESSION VECTOR OF SEROTONIN N-ACETYLTRANSFERASE GENE
- VernacularTitle: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
- Author:
Hua LIAO
- Publication Type:Journal Article
- Keywords:
The Serotonin-N-acetyltransferase(AA-NAT);
L6 myoblast;
Transfection pTARGET~(TM) mammalian expression vector system;
RT-PCR
- From:
Acta Anatomica Sinica
2002;0(06):-
- CountryChina
- Language:Chinese
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Abstract:
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 cDNA 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 eukaryotic expression vector pTARGET~(TM)-AANAT was transfected into rat L6 myoblasts with lipofectamine.Accordingly,engineered cells selected by antibiotic G418 were detected by the methods of RT-PCR and Westem blotting. Results It was revealed that,amplified AA-NAT cDNA confirmed by agarose gel electrophoresis could ligate with pTARGET~(TM) vector and subcloned into JM109 cells.L6 cells transfected with pTARGET~(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 eukaryotic expression system successfully although it was just a primary result.This system can be used for the transfection of L6 myoblast.
