檢測及改進能表現兩種抑菌蛋白之枯草桿菌轉殖品系的抗菌效力

Abstract

由於抗生素殘留對人體健康的疑慮，和抗藥性病原菌的產生及擴散，若能使用具有抗生素抗菌能力但不具有抗生素缺點的抗菌蛋白質，必能減少抗生素的使用，這在水產養殖與畜牧業上會有極大的助益。過去的實驗中，以安全微生物枯草桿菌(Bacillus subtilis)為宿主細胞，用質體轉殖的方式表現外源性抗菌胜肽Lactoferricin被證明是可行的。在這裡，我們以兩種方向來嘗試改進原來的表現載體以增進轉殖株的抗菌能力：其一是將質體攜帶的抗菌蛋白改為殺菌環境較廣的 Ciona molecule against microbes (CiMAM)；其二是將質體上表現抗菌蛋白的啟動子改為較強勢的PtrnQ。用電穿孔的方式，將表現質體轉殖進入細胞。轉殖後使用能夾取質體DNA中報導基因的引子(primers)進行PCR初步的篩選，分別挑選了170顆與480顆轉殖株；之後再以西方浸漬法(Western Blot)偵測CiMAM重組蛋白和lactoferricin重組蛋白的表現(在37 kDa與44 kDa處可偵測到訊號)，最後挑選到表現量佳及穩定性高的第一種轉殖株C117和C166與第二種轉殖株P245和P263。在in vitro的抑菌測試裡，我們先選常見的格蘭氏陰性的Escherichia coli (E. coli)和陽性病原菌的Staphylococcus epidermidis (S. epidermidis)為對象，結果得到5 ml之C117 (1.45 x 108 CFU·ml-1)、C166 (2.17 x 108 CFU·ml-1)與P245 (1.7 x 108 CFU·ml-1)、P263 (1 x 108 CFU·ml-1)之5.5 ml萃取液中，取其1 l的抑菌效果在E. coli中分別相當於87.43 ng、70.24 ng 與238.25 ng、322.7 ng的Ampicillin；在S. epidermidis中則相當於47.97 ng、40.5 ng 與366.4 ng、452.52 ng的Ampicillin。同時，我們也選擇了養殖漁業常見的淡水病原菌Edwardsiella tarda (E. tarda)試驗，結果得到以相同方式處理的C117、C166與P245、P263其 1 l萃取液的抑菌效果在E. tarda中分別為38.59 ng、37.47 ng 與168.04 ng、249.94 ng的Ampicillin。由於CiMAM來自生長在海洋環境中的玻璃海鞘(Ciona intestinalis)，因此又選擇了嗜鹽性細菌Vibrio natriegens (V. natriegens)進行測試，結果顯示C117、C166與P245、P263的1 l萃取液抑菌效果分別相當於57.06 ng、32.35 ng 與25.38 ng、32.93 ng的Tetracycline。若對海洋性病原菌Vibrio parahaemolyticus (V. parahaemolyticus)、Vibrio alginolyticus (V. alginolyticus)與Streptococcus iniae (S. iniae)進行測試，結果得到C117、C166與P245、P263之1 l萃取液的抑菌效果在V. parahaemolyticus中分別為47.07 ng、25.20 ng 與178.42 ng、192.81 ng的Tetracycline；在V. alginolyticus中分別為58.17 ng、36.55 ng 與264.25 ng、275.73 ng的Tetracycline；在S. iniae中分別為50.97 ng、39.28 ng 與219.7 ng、252.43 ng Tetracycline。最後，我們用對Ampicillin有抗藥性的E. coli進行測試，結果得到C117、C166與P245、P263之1 l萃取液的抑菌效果分別為42.77 ng、30.35 ng與250.39 ng、276.76 ng的Tetracycline。綜合上述研究結果證實，以枯草桿菌作為宿主細胞，可以表現外源性耐鹽性的抗菌蛋白CiMAM，其對海洋性細菌都具有比lactoferricin好的殺菌效果；另一方面，以PtrnQ為啟動子的轉殖株，和以前使用的P43啟動子並含有相近plasmid copy number的轉殖株相比較，擁有顯著的殺菌效力提升。這些轉殖株預期會有運用在養殖及畜牧業的潛力。

With the concerns about antibiotic residues on human health and the production and spread of drug-resistant pathogens, the use of antimicrobial peptides (AMPs) that have the bactericidal activity of antibiotics but do not have the shortcomings of antibiotics might reduce the widespread usage of antibiotics, which in turn, beneficial for aquaculture and animal husbandry. It has been reported that a biosafe probiotic microorganism Bacillus subtilis is a good host cell to express the exogenous AMPs such as lactoferricin (LFB) through gene transfer. However, not only the condition that LFB enables to present the antibacterial ability is relatively narrow but also the yield production of recombinant LFB is low. To solve these issues, in this study, we attempted to improve the transgenic strain of B. subtilis to enhance the antibacterial capability and production. In the first part, we replaced LFB by Ciona molecule against microbes (CiMAM) since CiMAM is isolated from Ciona intestinalis which habitats in the seawater so that it is more capable to kill bacteria at salt environment. After the expression plasmid containing CiMAM was constructed, we employed electroporation to introduce this plasmid harbored by the competent host cells, followed by PCR to screen 170 transformants. We further used Western blot analysis to examine the transgenic strains that enable to steadily produce recombinant CiMAM (rCiMAM) presented two positive signals localized at 37 and 48 kDa positions. Among them, two transgenic strains C117 and C166 were selected since they expressed rCiMAM in a stronger and more stable manner. In vitro bactericidal assay, results demonstrated that the bactericidal activities of 1 l aliquot of a total 5.5-ml extracted from 5-ml cultured C117 (1.45 x 108 CFU·ml-1) and C166 (2.17 x 108 CFU·ml-1) were equivalent to the efficacy of 87.43 and 70.24 ng of Ampicillin against Escherichia coli, respectively, while the bactericidal activities equivalent to the efficacy of 47.97 and 40.5 ng of Ampicillin against Staphylococus epidermidis, 38.59 and 37.47 ng of Ampicillin against Edwardsiella tarda, a common pathogen in aquaculture. Importantly, the bactericidal activities of 1 l aliquot extracted from C117 and C166 were equivalent to the efficacy of 57.06 and 32.35 ng of Tetracycline against Vibrio natriegens, a halophilic bacterium, while they were equivalent to the efficacy of 47.07 and 25.2 ng of Tetracycline against V. parahaemolyticus and 58.17 and 36.55 ng of Tetracycline against V. alginolyticus, suggesting that transgenic strains C117 and C166 expressing rCiMAM does display a higher bactericidal capability against halophilic bacteria than strains expressing LFB generated previously. Moreover, we also found that the bactericidal activities of C117 and C166 were equivalent to the efficacy of 50.97 and 39.28 ng of Tetracycline against Streptococcus iniae and 42.77 and 30.35 ng of Tetracycline against an Ampicillin- resistance E. coli strain. In the second part, we replaced a common P43 promoter by a stronger promoter PtrnQ, which is a promoter of tRNA gene, trnQ, within the expression plasmid to enhance the amount of recombinant LFB (rLFB) produced by host cell B. subtilis. Following the same strategy described abpve, we selected two transgenic strains P245 and P263 after screening from 480 transformants. In vitro bactericidal assay, results demonstrated that the bactericidal activities of 1 ul aliquot of a total 5.5-ml extracted from 5-ml cultured P245 (1.7 x 108 CFU·ml-1) and P263 (1 x 108 CFU·ml-1) were equivalent to the efficacy of 238.25 and 322.7 ng of Ampicillin against Escherichia coli, respectively, while the bactericidal activities equivalent to the efficacy of 366.4 and 452.52 ng of Ampicillin against Staphylococus epidermidis, 168.04 and 249.94 ng of Ampicillin against Edwardsiella tarda, a common pathogen in aquaculture. Likewise, the bactericidal activities of 1 l aliquot extracted from P245 and P263 were equivalent to the efficacy of 25.38 and 32.93 ng of Tetracycline against Vibrio natriegens, a halophilic bacterium, while they were equivalent to the efficacy of 178.42 and 192.81 ng of Tetracycline against V. parahaemolyticus and 264.25 and 275.73 ng of Tetracycline against V. alginolyticus. Furthermore, we also found that the bactericidal activities of P245 and P263 were equivalent to the efficacy of 219.7 and 252.43 ng of Tetracycline against Streptococcus iniae and 250.39 and 276.76 ng of Tetracycline against an Ampicillin-resistance E. coli strain. Based on this collective evidence, we demonstrated that the yield of rLFB driven by PtrnQ was greater than that of rLFB driven by P43 in the transgenic strains harboring the same number (around 1000 per cell) of plasmids. Taken together, we strongly suggest that the transgenic B. subtilis strains generated by this study are highly potential to be used in aquaculture and animal husbandry to prevent animals from bacterial infection during disease outbreak.