Expression of Human Cathelicidin LL-37 in Bifidobacterium longum and Determination of Its Activity

WANG Xiaokang, WU Tiesong, LIU Jianghong, XIE Zhanxiong*

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主办：上海医药工业研究院
　　　中国药学会
　　　中国化学制药工业协会
ISSN 1001-8255   CN 31-1243/R   ZYGZEA
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Chinese Journal of Pharmaceuticals
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Chinese Journal of Pharmaceuticals ›› 2018, Vol. 49 ›› Issue (01) : 68. DOI: 10.16522/j.cnki.cjph.2018.01.009
Paper

Expression of Human Cathelicidin LL-37 in Bifidobacterium longum and Determination of Its Activity

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Abstract

LL-37 is an oligopeptide possessing a broad-spectrum antibacterial ability. In the present work, the food-grade microbe Bifidobacterium longum (B. longum) was engineered for the secretion of active LL-37 and its antimicrobial activity as well as endotoxins neutralizing capacity of recombinant LL-37 were measured. For the construction of a secreted expression plasmid, a 149 bp hup promoter and a 150 bp amyB signal peptide were amplified by PCR from B. longum, and then they were cloned into the pBluescript II SK(-) vector together with a DNA fragment (pMB1) containing a replicon of B. longum and a synthesized 133 bp DNA of LL-37. The plasmid, named as pBs- LL-37, was transformed into B. longum by electroporation and the positive clone was identified by PCR. The obtained genetically modified strain was cultivated in liquid DMEM medium. Tricine SDS-PAGE result showed that recombinant LL-37 peptide (Mr 4 500) could be detected in the culture supernatant. The LL-37 concentration in the supernatant was (77.36±4.61)μg/ml after cultivation for 30 h. Agarose diffusion assay showed that the inhibition activity of recombinant LL-37 against Escherichia coli K99 was comparable with that of the synthesized LL-37. In the anti-endotoxin test, the recombinant LL-37 could decrease the TNF-α expression level of RAW264.7 cells induced by lipopolysaccharide (LPS) as with the synthesized LL-37. There were no significant differences between them. This work may provide some references for the research and development of recombinant LL-37.

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LL-37 / antibiotic peptide / Bifidobacterium / transgene / secreted expression

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WANG Xiaokang, WU Tiesong, LIU Jianghong, XIE Zhanxiong*. Expression of Human Cathelicidin LL-37 in Bifidobacterium longum and Determination of Its Activity. Chinese Journal of Pharmaceuticals. 2018, 49(01): 68 https://doi.org/10.16522/j.cnki.cjph.2018.01.009

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