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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2016, Vol. 36 Issue (8): 23-30    DOI: 10.13523/j.cb.20160804
    
The Fusion Expression of Bovine Lactoferricin (LfcinB) in Bacillus thuringiensis
LIU Jing-ying, LIU Cheng-lang, ZHANG Yun-lei, WANG Shi-qi, XIA Li-qiu, ZHANG You-ming
State Key Laboratory Breeding Base of Molecular Microbial Biology, College of Life Science, Hunan Normal University, Changsha 410081, China
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Abstract  

LfcinB is a small piece of peptide derived from bovine lactoferrin. It was reported that antibacterial activity of LfcinB stands its leading place among all kinds of lactoferricin. Preliminary research showed that, activated LfcinB can be expressed through E.coli expression system and Pichia pastoris expression system, but with limited yield and difficulty in purification. So building a new LfcinB expression system is significative. Baciullus thuringiensis can form ICPs consisting of plenty of δ-endotoxins during spore formation. It was subjected to lysis after fermentation,releasing spore and crystal into culture medium. Based on the above advantages of Bt, a fusion protein of LfcinB and Cry60Ba about 35kDa was constructed. The fusion gene was constructed by fusing the gene encoding LfcinB to the 3' terminus of the intact cry60Ba gene. The recombinant vector containing the fusion gene was transformed into acrystalliferous Baciullus thuringiensis strain 4Q7 recombinant strain 4Q7/pPFT60Ba-LfcinB was obtained. After cultivated in GYS medium for 48 h, the recombinant strain produced parasporal crystal, which was collected and subjected to SDS-PAGE analysis. The result showed that the expected protein band about 38 kDa was obtained. The purified fusion protein was hydrolyzed with HCl and subjected to SDS-PAGE analysis. The result showed that 3kDa target protein was obtained, which was then excised and was subjected to LC-MS/MS analysis. The result showeds that Cry60Ba and LfcinB from fusion protein was separated after hydrolysis and single LfcinB was obtained.This indicated that expressing fusion protein of crystal protein and LfcinB in Baciullus thuringiensis may be a efficient expression system of LfcinB, which laid the foundation for massively producting active LfcinB through genetic engineering methods.

Key words： Cry60Ba      Fusion expression      Baciullus thuringiensis      LfcinB     
Received: 27 January 2016      Published: 25 August 2016
ZTFLH:  Q789  
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LIU Jing-ying, LIU Cheng-lang, ZHANG Yun-lei, WANG Shi-qi, XIA Li-qiu, ZHANG You-ming. The Fusion Expression of Bovine Lactoferricin (LfcinB) in Bacillus thuringiensis. China Biotechnology, 2016, 36(8): 23-30.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20160804     OR     https://manu60.magtech.com.cn/biotech/Y2016/V36/I8/23

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