Study On Expression Of Human Obese Gene In E.coli

Leptin is the expression product of obese gene in adipose tissue, and it is reflected in body fat composition and body fat content and body weight regulation as an important signal factor.This study designed primers according to related literature, and after the PCR procedures with the annealing temperature of 68℃and 30 cycles, the leptin gene fragment of about 460bp in the plasmid vector was cloned. Then the target fragment was recoveried from the agarose gel, and cloned into the pMD18-T sequencing vector for sequencing analysis.After the identification with the correct sequence of target fragment, we digested the recombinant sequencing vector pMD18-T-OB with two enzymes directly, and recoveried the gene fragment of about 460bp. Then connected the leptin gene fragment and the pET-28a(+) expression vector with the same two enzymes digestion. The product was transformed into competent E. coli cell, and selected the colony grew on the resistant bacteria plate and vaccinated it into liquid culture. The plasmid was extracted and identificated with two enzymes digestion, and the recombinant plasmid was successfully constructed.The recombinant expression plasmid pET-28a(+)-OB was transformed into E. coli DH5αand BL21(DE3) strains, then they were induced with different IPTG concentrations and culture times. Then the bacterial protein was analysised by SDS-PAGE to identify if the recombinant gene was expressed. The results showed that there was a large amount of leptin expression in the BL21 (DE3) strains, but for the DH5αstrains, there was no protein of expected molecule weight (for leptin, it is about 16700Da) could be detected. The results indicated that the E. coli BL21(DE3)(pET-28a(+)-OB) genetic engineering strains which could express leptin in large amount was successfully constructed.