Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (37): 6926-6930.doi: 10.3969/j.issn.2095-4344.2012.37.018

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Construction of a recombinant pET-32a-leptin efficient expression vector

Xie Lin1, Wu Guo-hui2, Li Xiao-lin2, Wen Hui-cai3   

  1. 1Department of Ophthalmology, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangsu Province, China
    2Department of Plastic & Dentofacial Surgery, Jiangxi Provincial People's Hospital, Nanchang 330006, Jiangxi Province, China
    3Department of Plastic & Aesthetic Surgery, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China
  • Received:2012-04-02 Revised:2012-07-08 Online:2012-09-09 Published:2012-09-09
  • Contact: Wu Guo-hui, Master, Associate chief physician, Master’s supervisor, Department of Plastic & Dentofacial Surgery, Jiangxi Provincial People's Hospital, Nanchang 330006, Jiangxi Province, China wu_gh@163.com
  • About author:Xie Lin☆, Doctor, Attending physician, Department of Ophthalmology, the Second Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangsu Province, China amandaxie0510@yahoo.com.cn

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Abstract:

BACKGROUND: Leptin is recognized as a feedback signal of body fat by bonding receptor in the hypothalamus, which is related to ingestion, drinking, and energy metabolism.
OBJECTIVE: To construct a prokaryotic recombinant expression plasmid pET-32a-leptin and to analyze its expression in BL21 Escherichia coli.
METHODS: The adipose tissues were obtained, and leptin gene was obtained by reverse transcription-PCR. The target gene was orientating cloned into pMD18T and pET-32a vector by DNA recombinant technical. Position clones were transformed into BL21 Escherichia coli and expressed by double digestion and DNA sequencing. Then the expression product was detected for the antigen reactionogenicity after degeneration and renaturation.
RESULTS AND CONCLUSION: 520 bp leptin fragment was amplified and the prokaryotic recombinant expression plasmid pET-32a-leptin was correctly constructed. The Leptin gene fragment in position clones was tested be same as the sequence of the GenBank public by DNA sequencing. The position protein pET-32a-leptin was highly expressed and about 50% on total protein. The antigen reactionogenicity of expression product was enhanced after degeneration and renaturation (P < 0.01). Prokaryotic recombinant expression plasmid pET-32a-leptin was successfully constructed and the target protein was expressed largely.

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