RESUMEN
Objective To identify the sequence of cDNA of human leptin coding region, construct a prokaryotic expression vector, and express human leptin in E.coli. Methods RNA was extracted from human placenta tissue. Leptin gene was amplified from RNA by RT-PCR method. The PCR product was ligated with T vector. The ligation reaction was transformed to E.coli DH5? competent cells. The recombinant plasmid was checked by sequencing and restriction analysis. The human leptin gene was cloned into prokaryotic expression vector PET-28a and tranformed into E.coli BL21. The recombinant strain was constructed and induced by IPTG. The product was analyzed with SDS-PAGE and Western blotting. The expressive product was purified by sodium deoxycholate. Results Analysis indicated that the sequence of human leptin cDNA was the same with the reported sequence. Leptin gene was inserted into prokaryotic expression vector. The fusion protein expressed with high efficiency in recombinant E.coli BL21.The results of SDS-PAGE analysis indicated that the molecular weight of the fusion protein was about 20kD. Conclusion Hunam leptin gene was successfully identified from placenta. The leptin gene prokaryotic expression strain was constructed and a high expression of leptin was achieved in E.coli.