[The construction and the expression of V5 epitope fused human androgen receptor vector in the yeast cell].
When we try to establish the gene recombinant yeast cell to screen the androgenic endocrine disruptors, the key procedure is the androgen receptor (AR) expression in the yeast cell. For this purpose, we obtained the GPD (glyceraldehyde-3-phosphote dehydrogenase) promoter from the yeast genosome of W303-1A using PCR system and inserting it into Swa I and BamH I sites of pYestrp2. The new constructed vector was named pGPD. The V5 epitope tag DNA with a 5'-BamH I and a 3'-EcoR I sticky end was cloned into the corresponding site of the pGPD vector to yield the vector of pGPDV5. The 2 723 bp full length AR ORF amplified by PCR from pcDNA3.1/AR was fused to V5 epitope tag DNA in pGPDV5 to give the AR yeast expression vector of pGPDV5/AR. This fused vector was transformed into the yeast cell (W303-1A). Western blot was used to detect the V5 fused protein of AR, in the protocol of which the primary monoclonal antibody (IgG(2a)) of mouse anti-V5 and the polyclonal secondary antibody of goat anti-mouse (IgG) linked to horseradish peroxidase (HRP) were used to detect the specific protein in the given sample of the transformed yeast extract. The result showed that the fused protein of AR was expressed successfully in the yeast cell.
Department of Preventive Medicine, Medical College, Yangzhou University, Yangzhou 225001, China.
Chen
C
Yang
Fangni
F
Luo
Weixing
W
Dai
Shanshan
S
Li
Renhua
R
Huang
Yangmei
Y
Xie
Feiyue
F
Xue
Xiangming
X
Li
D001483:Base Sequence; D052244:Endocrine Disruptors / Q000032:analysis; D000939:Epitopes / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011944:Receptors, Androgen / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D052244:Endocrine Disruptors; D000939:Epitopes; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases