Gene Of Human Adiponectin Cloning By PCR-driven Overlap Extension And Expression In Pichia Pastoris

In this research,gene of human adiponectin was cloned by PCR-driven overlap extension.Then the cloning vector pGEM-T-ADPN was constructed.After the sequence was determined,the ADPN gene was subcloned into the expression vector pPIC3.5K to yield the recombinant expression vector pPIC3.5K-ADPN.The recombinant plasmid was transformed into P.pastoris GS115 by electroporation. After induction by methanol,ADPN was expressed in GS115.The main achievements of this research were as follows:The second and third exons of human ADPN gene were amplified from human blood by PCR-driven overlap extension.The ADPN gene was ligated into the pGEMT vector.The recombinant vectors were assayed by restriction digestion and sequencing.Then the sequence was blasted with the human ADPN cDNA titled NM₀04797.2 in the NCBI.And the homology was 100%.The results showed that the ADPN gene was successfully cloned and ligated into the cloning vector pGEM-T.For constructing the expression vector pGEM-T-ADPN,two primers of ADPN gene with BamHâ… and EcoRâ… site respectively were designed.Then the ADPN gene with the two restriction enzyme sites at its 5' end and 3' end respectively was amplified and inserted into the expression vector pPIC3.5K.The recombinant expression vector pPIC3.5K-ADPN was transformed into DH5αand identified by PCR and restriction enzyme BamHâ… and EcoRâ… .The results indicated that the gene was correctly cloned into the vector.The expression vector pPIC3.5K-ADPN was transformed into P.pastoris(GS115) by electroporation.Many recombinants were obtained after a series of screening, including auxotroph,G418,PCR and Southern blotting.After induced by 1.0% methanol,human ADPN was expressed in GS115.By the western blotting analysis,the optimum conditions of expression are 30℃,1%methanol inducing 48 h.The results indicated that the human ADPN protein expressed succesfully in GS115 and the protein has strong antigenicity.