Expression Of Recombinant Human GAdiponectin In E.coli BL21 (DE3) And Pichia Pastoris

Adiponectin is a kind of important active molecules produced by adipocytes, which is closely linked obesity and type 2 diabetes and cardioascular disease in the blood circulation. Studies have shown that gAdiponectin have the same biological effect to these diseases.Therefore, it could be developed a new drug to apply to treat diabetes in the future.In this study, we constructed prokaryotic and eukaryotic expression vectors for gAdiponectin gene. The objective is to comparative study expression of gAdiponectin protein in E.coli BL21(DE3) and Pichia pastoris.Designing His-Tag primers with restriction sites of EcoR I and Not I, we got fusion gene by PCR strategy, which is named His-gAd.â‘ The fusion gene was connected with a prokaryotic express vector pET32a(+), and then transformed into E.coli DH5a. After Screening and analyzing the positive strains, we cloned recombinant vectors into E.coli BL21(DE3) and got the gAdiponetin protein in recombinant prokaryotic strains.â‘¡We constructed eukaryotic express vectors pPIC9k-His-gAd, which were transformed into E.coli DH5a and Pichia pastoris. The transformants were screened and induced to express the gAdiponetin protein by 0.5% methanol.â‘¢After analyzed by the methods of SDS-PAGE and Western Blot, the protein product was collected after purification by His-Binding-resin column and ultrafiltration. In the end, we measured the levels of the gAdiponetin protein by Bradford method.Conclusions:The recombinant strains were assayed by sequencing. Then the sequence was blasted with the human gAd cDNA titled NM₀04797.3 in the NCBI. And the homology was 100%. The results showed that the gAd gene was successfully cloned and ligated into the expression vector.The target strips can be saw obviously after analysis by SDS-PAGE and Western Blot. The respective levels of the gAdiponetin protein were 56.6mg/L and 13.5mg/L fermentation liquid.