关键词: 高原鼠兔, 瘦素原核表达, 蛋白纯化

Abstract: Leptin,the production of the ob gene,plays an important role in the regulation of energy homeostasis. Plateau
pika (Ochotona curzoniae) leptin is closed to cold tolerance. In this study,we establish an effective method for expression of recombinant pika leptin in Escherichia coli. The gene sequence encoding pika leptin was obtained by PCR from the plateau pika cDNA library and the PCR product was cloned into pET30a(+ )by DNA recombination techniques. After DNA sequencing, the confirmed recombinant clone pET30a(+ )/ ppleptin was transformed into BL21(DE3)for expression under the induction of IPTG. Due to the expressed protein,the insoluble inclusion body must be separated,denaturated and purified with a Ni sepharose column. The sequencing results of pET30a(+ )/ ppleptin vector demonstrated that the insert of the pika leptin gene was the same as that of pika gene in GeneBank. At the same time the recombinant protein was identified by SDS - PAGE,and the results revealed that there was a new band of protein around 16KD;this protein was purified successfully. Our results showed that the prokaryotic expression system of pika leptin has been successfully constructed and the purified recombinant protein provides a basis for further research of plateau pika leptin.

Key words: Plateau pika (Ochotona curzoniae), Leptin prokaryotic expression, Protein purification