Cloning And Eukaryotic Expression Of Shaziling Porcine Adiponectin Gene

As an adipocytokins secreted exclusively by adipocyte, adiponectin has many biological functions within animal body, such as reducing lipid accumulation and increasing fatty acid oxidation resisting inflammatory reaction and injury of blood vessel. It may play important roles in regulating energy homeostasis,glucose and lipid metabolism especially. In this paper, the source,structure, biological functions of adiponectin and the factors which may effect adiponectin secretory, were introduced. The effects of adiponectin on animal fatty deposition were summarized. And the prospects of adiponectin utilizing in pig production were also discussed.Pichia pastoris protein expression system has developed a eukaryotic expression system in recent years. The value of its research and application is very extensive for its many advantages compared with other expression systems. At present, Pichia pastoris protein expression system has successfully express a variety of proteins. In the present study we designed to get a high yield of active recombinant adiponectin in pichia pastoris GS115.The experimental section of this study includes the chapter of Shaziling porcine adiponectin gene clone and the chapter of expression in Pichia pastoris.In experiment l,we extracted the total RNA from the Shaziling porcine adipose tissue and got the adiponectin cDNA with the RT-PCR method and a couple of designed and specific primers(ADPN-Pl and ADPN-P2).The recombinant expression vector that was named as pMD-ADPN was transformed into the coli.DH5a for sequencing.The sequencing results showed that the cloned adiponectin gene sequence length was732bp and encoded proteins of244amino acid residues. After NCBI BLAST,it showed the amino acid sequence of adiponectin had100%homology compared to porcine adiponectin have been published in genbank.lt fully proves that Shaziling porcine adiponectin gene has been cloned successfully in the present study.In experiment2, the cloned adiponectin cDNA have been integrated into the expression vector pPICZaA to construct the recombinant expression vector pPICZaA-ADPN. pPICZaA-ADPN was transformed into Pichia pastoris GS115by electroporation,and that the recombiantant Pichia pastoris was named as GS115-ADPN.After the induction of methanol, adiponectin gene was expressed in the culture medium of the GS115-ADPN.We collected target protein and made Western-Blot appraisal.The results showed that the recombinant adiponectin have been expressed correctly in Pichia pastoris GS115and had bioactivity. Also, Bradford appraisal was utilized to measure that the level of constructed GS115-ADPN expressing in adiponectin protein is28.5μ g/mL.