利用枯草桿菌大量表現具有抗菌能力的乳鐵蛋白

Using Bacillus subtilis as a Host Cell to Express Recombinant Bactericidal Protein

洪浚瑋(Jun-Wei Hung)

指導教授 ： 蔡懷楨

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202001884

摘要

為了開發出水產養殖業和畜牧業常用抗生素的替代品，我們使用安全性微生物Bacillus subtilis去建立一個表現外源性抗菌蛋白(lactoferricin)的轉殖株。將構築好的質體(pP43-YncM-6LFBII-mdGFP)以電穿孔的方式轉殖進B. subtilis之competent cells，使用夾取lactoferricin cDNA的primers對轉殖株的colony進行 PCR，初步篩選得到含有lactoferricin cDNA片段的轉殖株T7、T13、T1以及T4，再將這些轉殖株進行Western blot analysis偵測recombinant lactoferricin蛋白質的表現，再進一步偵測質體的穩定性。經過約20個世代後，只剩T1和T13留有穩定的質體。利用dot blot analysis偵測這兩種轉殖株的質體，發現每顆T1細菌帶有931個質體；而每顆T13細菌帶有647個質體。在in vitro的抑菌實驗中，我們選擇一般常見的革蘭性陰氏菌Escherichia coli和革蘭性陽氏Staphylococcus epidermidis進行抑菌測試。結果顯示，在E. coli抑菌實驗中，轉殖株T1、T13萃取液中取1 ul (T1:4.2 x 105 CFU, T13:3 x 105 CFU)的抑菌效果分別相當於56 ng、53 ng Ampicillin；在S. epidermidis抑菌實驗中，轉殖株T1及T13萃取液中取1 ul的抑菌效果分別相當於154 ng及130 ng的Ampicillin。另外，在養殖漁業常面臨的一些腸道感染菌，包括Vibrio parahaemolyticus和Edwardsiella tarda在抑菌測試中也達到相當的效果。另外，為了想要更有效率地表現出recombinant lactoferricin，我們使用了比P43 promoter更強勢的PtrnQ promoter來驅動recombinant lactoferricin cDNA，藉此表現更多的recombinant lactoferricin來達到更好的抑菌效果。我們也了解轉殖株的穩定性可能成為其潛在的危機，並使用CRISPR/Cas9 system將recombinant lactoferricin cDNA嵌插至B. subtilis的chromosome中，得到穩定的轉殖株。本研究證實將安全性微生物B. subtilis作為宿主，可以穩定表現外源性抗菌蛋白且抑菌效果顯著，預期之後可以應用於水產養殖業和畜牧業以減少疾病造成的損失。

關鍵字

枯草桿菌 ； 乳鐵蛋白

並列摘要

To develop an alternative to conventional antibiotics used in the aquaculture and livestock industries, we employed Bacillus subtilis, considered a safe microorganism, to express the exogenous antimicrobial peptide lactoferricin. We constructed an expression plasmid pP43-6LFBII-mdGFP, which was electroporated into B. subtilis competent cells, followed by regeneration and cultivation. The putative colonies harboring the transferred plasmid were primarily screened by PCR-amplification of lactoferricin cDNA fragment. Four transformants possibly containing lactoferricin cDNA included strains T7, T13, T1 and T4. Based on Western blot and dot blot analyses, we found that transgenic strains T1 and T13 not only expressed exogenous recombinant lactoferricin, but also exhibited stable inheritance of plasmids with 931 and 647 copies per cell, respectively. In the antibacterial in vitro experiment, the bactericidal activity of each microliter of cell lysate from transgenic strains T1(4.2 x 105 CFU) and T13 (3 x 105 CFU) for Gram-negative Escherichia coli was equivalent to 56 and 53 ng of Ampicillin dosage, respectively, while for Gram-positive Staphylococcus epidermidis, the equivalency T1 and T13 were 154 and 130 ng of Ampicillin dosage, respectively. Equivalencies of bacterial activity for Vibrio parahaemolyticus and Edwardsiella tarda followed suit. In addition, in order to express recombinant lactoferricin more efficiently, we choose a stronger PtrnQ promoter than the P43 promoter to drive the recombinant lactoferricin cDNA and express more recombinant lactoferricin to achieve better bactericidal activity. We realize the stability of the transgenic strains may be a potential crisis, hence we use the CRISPR / Cas9 system to insert the recombinant lactoferricin cDNA into the chromosomes of B. subtilis, therefore, stable transgenic strains were obtained. This study confirms that using the safe microorganism B. subtilis as host cells can stably express exogenous antimicrobial proteins with significant bactericidal activity. It is expected that it can be applied to aquaculture and animal husbandry to reduce losses caused by disease in the future.

並列關鍵字

Bacillus subtilis ； lactoferricin

參考文獻

Aguilera. O, Ostolaza. H, Quirós. L.M, Fierro. J.F, Permeabilizing action of an antimicrobial lactoferricin-derived peptide on bacterial and artificial membranes, FEBS Lett 462(3) (1999) 273-277.

Actor. J.K, Hwang. S.A, Kruzel. M.L, Lactoferrin as a natural immune modulator, Curr Pharm Des 15(17) (2009) 1956-1973.

Bellamy. W, Takase. M, Yamauchi. K, Wakabayashi. H, Kawase. K, Tomita. M, Identification of the bactericidal domain of lactoferrin, Biochim Biophys Acta Protein Struct Mol 1211(1-2) (1992) 130-136.

Bellamy. W, Takase. M, Wakabayashi. H, Kawase. K, Tomita. M, Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin, J Appl Bacteriol 73(6) (1992) 472-479.

Brockmeier. U, Caspers. M, Freudl. R, Jockwer. A, Noll. T, Eggert. T, Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria, J Mol Biol 362(3) (2006) 393-402.