Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66358
標題: LL-37融合蛋白與化學合成LL-37以及生化合成LL-37之生產效益和抗菌功能的評估
Evaluation of production performance and antimicrobial function of the LL-37 peptide as fusion protein and as chemically synthesized and biologically generated peptide
作者: 劉恆仲
CHUNG, LIU HENG
關鍵字: Antimicrobial peptide;抗菌蛋白
出版社: 獸醫微生物學研究所
摘要: 
中文摘要
本實驗在評估化學合成的LL-37抗菌胜 (peptide) 和生化合成的LL-37融合蛋白的抗菌功能和生產效益。當人類上皮細胞受到微生物侵入引起局部性發炎時,會產生各種成份的胜,以消滅外來入侵的細菌。其中之一的基因是以豬的PR-39之cDNA作為探針,自人類骨髓cDNA基因庫分離到,此基因的蛋白產物由約170個氨基酸組成,它可被切割成二個片段,N端為cathelin propart,是一種cysteine protein inhibitor,C端為LL-37 antimicrobial peptide,以經過純化的LL-37,進行細菌抑制環分析(inhibition zone assay),顯示其具有抗菌性。它會形成a-helix的結構,且能溶解細菌細胞膜。利用E. coli量產LL-37抗菌胜,將此基因序列插入由125個氨基酸組成的 ketosteroid isomerase (KSI) 下游,及一段His-tag序列的上游。在E. coli表現的KSI/LL-37蛋白質形成的包涵體,自膠體切割回收和分離後,以細菌抑制環分析測試,顯示對革蘭氏陽性及陰性菌均有抗菌活性,但MIC比文獻報導略高。文獻指出,環境因子影響抗菌胜的結構和相異分子間作用的干擾,必須適當調控。接著我們也成功地利用大腸桿菌,表現出一段含三種功能區的LL-37/CBD/RGD融合蛋白質,它可能也受環境影響和融合蛋白的其他部份影響,抗菌效果需進一步研究。

Abstract
The aim of this study is to evaluate production performance and biological function of the chemically synthesized LL-37 peptide and the LL-37 as biologically generated fusion protein. Upon infection by microbes, human beings produce antimicrobial peptides as a defense mechanism. One of such genes was isolated from a cDNA library of human myeloid cells by probing with PR-39 cDNA of swine. Protein product of this gene is composed of 170 amino acid residues. It could be subdivided into two fragments. The N-terminal cathelin propart is a cysteine protein inhibitor and the C-terminal LL-37 peptide is an antimicrobial peptide. Inhibition zone assay indicated that purified LL-37 shows antimicrobial activity. Having been demonstrated to form a-helical structure, it could solubilize bacterial membrane. In order to overproduce LL-37 peptide in Escherichia coli, its coding sequence was inserted downstream of a 125 amino acid ketosteroid isomerase (KSI) gene and upstream of a His-tag sequence. The KSI/LL37 overproduced as inclusion bodies was purified from SDS-PAGE gel slice. Inhibition zone assay revealed that it has antimicrobial activity against both gram-positive and gram-negative bacteria, although its MIC appears higher than what has been reported. It has been reported that antimicrobial activity of peptide is influenced by various molecules in the environment and needs to be well controlled. In addition, we also produced in E. coli a tripartite chimeric protein LL-37/CBD/RGD. Its antimicrobial activity, probably also influenced by other components of the chimeric protein, needs to be re-evaluated.
URI: http://hdl.handle.net/11455/66358
Appears in Collections:微生物暨公共衛生學研究所

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