Cloning And Expression Of Human Adiponectin Gene And Its Bioactivity Determination

In the past twenty years, the rate of overweight and obesity have increased epidemiologically both in the developed and developing countries .The incidence of type 2 diabetes, cardiovascular disease and hypertension increased relatively. The adipose tissue is now considered not only as an energy storage but as a very important endocrine organ. Adiponectin is the protein secreted exclusively by adipose tissue, it exerts multiple effects such as anti-diabetic, anti-insulin resistance and anti-atherosclerotic effects.The hypoadiponectinemia was correlated with insulin resistance and type 2 diabetes. We can find the plasma adiponectin level reduced in the condition of CVD, hypertension, and dyslipidemia. As well, the determination of plasma adiponectin has paramount importance just like the plasma HbA1c determintion. So the plasma adiponectin determination will be the good marker for diagnosis and prognosis of diabetic metabolism.Adiponectin has direct action on the liver, skeleton muscle, and it can enhance the insulin sensitivity. Adiponectin can be used as a good insulin sensitizer which has the same effects as the TZD. Recently, in vitro experiments showed that adiponectin can repress the gluconeogenesis of the hepatocytes by inhibiting the expression of glucose-6-phosphatase gene.Obsjectives: 1. To clone and express of adiponectin to get the pure recombinent adiponectin and the polyclonal antibody. 2. To investigate the effects of recombinent adiponectin on the transcription level of G6Pase in hepatocytes. Methods:1. To extract the total RNA from the adipose tissue in a woman underwent the hysteromyoma surgery. We got the adiponectin cDNA with the RT-PCR method andwas cloned in to the expression vector pET-DEST42, The recombinent expression vector was named as pET-DEST 42/adiponectin. The expression vector then was transformed into the DE3 competent cell, and protein expressed induced with IPTG. The protein in the inclusion body was extracted and purified in the denatured condition. Protein was refolded with NDSB201 combined with GSH. The purity and concentration of the protein was determined, and stored at -80 °C.2. The polyclonal antibody was prepared with immunization of the rabbit and antibody titer was determined with the gel double-diffusion method.3. Primary hepatocytes separated from the liver of the rat was cultured for 4 hours. Add the protein in the incubation solution, semi-quantitative RT-PCR was performed 24 hours later. We investigated the effect of the protein on the transcription level of G6Pase.Results: l.The expression vector was constructed successfully, and sequenced to ensure the right clone. 2. Pure protein refolded to have the active bioactivity and the polyclonal antibody prepared.Conclusions: The gateway expression system has the advantages to clone the expression vector quickly and correctly, but the vector selection is still the key to improve production level of pure protein. Recombinent adiponectin has bioactivity, it can reduce the transcription level of G6Pase to regress the gluconeogenesis incultured primary epatocytes.