| Resistin is a newly discovered hormone secreted by adipocyte, it was thought to be a factor which induce the insulin resistance, regulate glucose metabolism and inhibit fat forming. The found of Resistin provided a new hot spot for study the animal disease of energy metabolism disorder, especially for cow adiposis hepatica disease which has relationship to the insulin-resistance (IR). IR commonly exists in metabolic syndromes such as adipositas, diabetes and hyperlipemia etc. Liver, peripheric fat and muscle tissue have lower biological response to the insulin than optimum level, it shew compensatory hyperinsulinemia with multiple metabolic disorder emergency reaction. Concrete phenomena are that more peripheral fat were mobilized, more FFA were uptaked, but oxidated or utilizated reduced , and more TG were formed in liver, and the ability of hepatocytes transfering fat out of liver was damaged, and then more fat was accumulated in liver cells. In addition, , it further mediated metabolism and immune system dysfunction that the IR-related hormones such as leptin, estrogen, cortisol, growth hormone, glucagon increased , and promote the formation of fatty liver. Therefore, the study of resistance protein structure and function of cattle lay the foundation to the application of recombinant resistin protein to prevent fatty liver of cow.Resistin is a secretion protein with cysteine-rich found in the Plasma, belongs to the resistance-likemolecules (RELMs), RELMs family contain–ing 105~117 amino acids, divided into three regions: the N-terminal signal sequence, the not fixed middle part , the highly conserved cysteine with repeat sequences constitutes the unique C-terminal function region. According to bovine resistin gene sequence from Genbank, One pair of specific primers were designed in this experiment.Taking the calf omental adipose tissue RNA as a template,the resistin gene were amplified by RT-PCR,and then cloned to pMD18-T. The sequence is determined, after the plasmid was identified as positive by the PCR and the 2 enzymes digestion. The sequence analysis revealed that the gene amplified by the PCR was the same as the sequence in Genbank. Suggested 327bp bovine resistin gene which encoding 109 amino acids has been successfully cloned .Compare and analyze the homology of resistin gene from cattle, pigs and mice, it was found that cattle and pigs share the highest homology as 85.5%, compared with the human and mice , homology were 80.3% and 63.7% respectively.The formation of Disulfide bond may be one of the necessary steps to active resistin, resistin dimer with 80%～90% of all functional protein, however, resistin monomers have only 10%～20% function of the entire pretein. Therefore, the formation of disulfide bonds is very important to maintaining the space-conformation and biological activity of risistin. In order to prepare high biological activity of resistin pretein, we choose pMAL-p2x expression vector for the expression of bovine recombinant resistin protein. Resistin gene was cloned to the prokaryotic expression vector pMAL-p2x, then got a pMAL-Resistin recombinant expression vector, and expressed in Escherichia coli TB1. SDS-PAGE analysis revealed the successful expression of recombinant plasmids, which wasexpressed in the cytoplasmic with 54.6kDa, protein is 221.7ug/mL after purification.The activity of bovine recombinant resistin was detected by the effection of bovine recombinant resistin on HSLmRNA in adipocytes. The results showed that recombinant resistin protein can promote the expression of HSL gene and weaken effection of insulin on adipocyte HSL expression, it indicates the recombinant resistin protein has the same biological activity as the nuture resistin.
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