( We provide with ABHD10 qPCR primers for gene expression analysis, HP103087 )
The pGEM-T is 3kb in length, and contains the amplicin resistance gene, conferring selection of the plasmid in E. coli, and the ori site which is the bacterial origin of replication. The plasmid has multiple cloning sites as shown below. The coding sequence was inserted by TA cloning. Many E. coli strains are suitable for the propagation of this vector including JM109, DH5α and TOP10.
The coding sequence can be easily obtained by digesting the vector with proper restriction enzyme(s). The coding sequence can also be amplified by PCR with M13 primers, or primer pair SP6 and T7.
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), C-GFPSpark 標籤||HG14439-ACG|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), C-OFPSpark 標籤||HG14439-ACR|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), N-GFPSpark 標籤||HG14439-ANG|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), N-OFPSpark 標籤||HG14439-ANR|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), C-Flag 標籤||HG14439-CF|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), C-His 標籤||HG14439-CH|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), C-Myc 標籤||HG14439-CM|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), C-HA 標籤||HG14439-CY|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), N-Flag 標籤||HG14439-NF|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), N-His 標籤||HG14439-NH|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), N-Myc 標籤||HG14439-NM|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體), N-HA 標籤||HG14439-NY|
|人 ABHD10 基因全長cDNA ORF克隆 (表達載體)||HG14439-UT|
Mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), is primarily metabolized by glucuronidation to a phenolic glucuronide (MPAG) and an acyl glucuronide (AcMPAG). It is known that AcMPAG, which may be an immunotoxic metabolite, is deglucuronidated in human liver. AcMPAG deglucuronidation activity was detected in both human liver cytosol (HLC) and microsomes (HLM). By purification from HLC with column chromatographic purification steps, the enzyme responsible for AcMPAG deglucuronidationis identified as α/β hydrolase domain containing 10 (ABHD10). Recombinant ABHD10 expressed in Sf9 cells efficiently deglucuronidated AcMPAG with a K(m) value of 100.7 ± 10.2 μM, which was similar to those in HLM, HLC, and human liver homogenates (HLH). Immunoblot analysis revealed ABHD10 protein expression in both HLC and HLM. The AcMPAG deglucuronidation by recombinant ABHD10, HLC, and HLH were potently inhibited by AgNO(3), CdCl(2), CuCl(2), PMSF, bis-p-nitrophenylphosphate, and DTNB. The CL(int) value of AcMPAG formation from MPA, which was catalyzed by human UGT2B7, in HLH was increased by 1.8-fold in the presence of PMSF. Thus, human ABHD10 would affect the formation of AcMPAG, the immunotoxic metabolite.