Abstract 819

Objective: The successful cloning of ob gene in 1994 is a breakthrough in the mechanisms of obesity. Ob gene is highly conserved among vertebrates and two classes of ob cDNA have been found which differ by inclusion and exclusion of a single glutamine codon at position +49. Leptin, the product of ob gene secreted primarily from adipocytes as a soluble hormone, plays important roles in regulating satiety, energy balance, neuroendocrine, sexual maturation and reproduction. Therefore, leptin has been considered to be an effective drug for the therapy of obesity. At present the ob gene has not been studied widely and there is not any report about the method of producing active recombinant human leptin in China, so this work has been done to obtain the sequence of Chinese OB cDNA and to establish a method of leptin production in China.

Methods: Chinese Han OB cDNA fragment was obtained by RT-PCR with total RNA extracted from human adipocytes and was inserted into the expressing vector pBV220. Then the constructed recombinant plasmid pBV220-OB was transformed to E.coli DH5 α for leptin expression. The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression. E.coli cells were lysed by high-pressure homogenization. After cell membrane extracted the inclusion bodies were mainly renatured and purified primarily by precipitation with ammonium sulfate and gel chromatography through a Sephadex G75 column. The activity of recombinant leptin was determined by its influence on the satiety and weight gain of mice.

Results: Analysis of DNA sequence showed that Chinese Han OB cDNA included the glutamine codon at +49. The amount of recombinant leptin expressed in E.coli accounted for 31-47% of total cellular proteins. From 1L of fermentative bacteria about 40mg of pure recombinant human leptin was isolated with a purity of being above 95%. After being administered to mice the recombinant human leptin could reduce their food intakes and inhibit weight gains.

Conclusions: The glutamine codon at 49 is not missing in Chinese OB gene. The biologically active human leptin can be obtained by relatively simple method of recombinant DNA technology.