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Gene cloning of porcine adiponectin gene from adipose tissue and construction of its eukaryotic expression vector
Author(s) -
Luo J.J.,
Song H.W.,
Zhang B.,
Li L.L.,
Chen Y.G.,
Peng Y.,
Wu L.Z.,
Fan J.X.,
Zhan J.S.
Publication year - 2014
Publication title -
journal of animal physiology and animal nutrition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.651
H-Index - 56
eISSN - 1439-0396
pISSN - 0931-2439
DOI - 10.1111/jpn.12101
Subject(s) - microbiology and biotechnology , expression vector , subcloning , pichia pastoris , recombinant dna , biology , gene , genetics
Summary To clone adiponectin (ADPN) gene from Shaziling porcine adipocyte and construct its eukaryotic expression vector, total RNA was extracted from subcutaneous fatty tissue. One pair of specific primers was designed by Primer 5.0 software according to the sequence of ADPN gene of porcine available in GenBank. The ADPN gene was amplified by PCR from cDNA and cloned into pMD 18‐T vector to construct recombinant clonal vector pMD ‐ADPN, sequenced and analysed. A recombinant expression plasmid pPICZ a A ‐ADPN was constructed by subcloning the cloned ADPN gene into the linearized pPICZ a A vector. Then, the plasmid pPICZ a A ‐ADPN was expressed in Pichia pastoris (GS115) by electrotransformation. Western blot and Bradford analysis were used to determine the target protein induced by methanol. Results showed that the genome size of ADPN was 732 bp and encoded 244 amino acid, the nucleotide sequence of ADPN shared 100% identity with that of porcine available in GenBank. Western blot and Bradford analysis showed that the recombinant ADPN was expressed in GS115 correctly and has certain immune activity. The expression level of ADPN was 28.5 μg/ml. In conclusion, the recombinant ADPN could express in eukaryotic expression vector pPICZ a A ‐ADPN constructed in this study effectively.