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Antibacterial Activity of Lactoferrin and Its Application in Detection of Mastitis
Chen, Po Wen
|摘要:||乳鐵蛋白為哺乳動物體內存有的天然抗菌蛋白。為解決畜牧現場的細菌抗藥性問題，所以進行乳鐵蛋白的抗菌功能及其應用之研究。首先，為了構築更強效的抗菌蛋白，利用疏水性和鹼性胺基酸與抗菌活性有關之概念，合成不同胺基酸序列的牛乳鐵蛋白衍生片段，研究構築更強效抗菌蛋白的可能性，並探討與抗菌活性有關的胺基酸序列。先合成牛乳鐵蛋白N端的20至29序列（bLf 20-29），並將其上所有的鹼性胺基酸以精胺酸或離胺酸替代；另將bLf 20-29上所有的非鹼性胺基酸以色胺酸、苯丙胺酸或異白胺酸替代。結果顯示，所合成的蛋白片段均有不等的抗菌活性，而精胺酸、離胺酸、色胺酸、苯丙胺酸和異白胺酸均與蛋白片段的抗菌活性有關。為了進一步產製抗菌蛋白以臨床運用，利用牛乳鐵蛋白素能抗高溫高壓滅菌而仍具抗菌活性之特點，建立了大腸桿菌表現系統以表現牛乳鐵蛋白素融合蛋白。欲待大量產製融合蛋白後，再經高溫高壓滅菌而直接應用於臨床。然而，所表現的融合蛋白本身及其經不同蛋白酶截切後的蛋白水解液均無抗菌活性。因此，另以容易取得之牛乳鐵蛋白為研究對象，研究牛乳鐵蛋白水解液與不同抗菌機制的抗生素併用後，對抗藥性大腸桿菌株的抗菌活性。結果發現，合併使用牛乳鐵蛋白水解液與gentamicin、cephalothin、cefamandole或rifampicin後，對抗藥性大腸桿菌菌株具有部分協同的抑制生長效果；而合併使用牛乳鐵蛋白水解液與rifampicin後，對抗藥性大腸桿菌株具有協同的殺菌效果。這些發現也許能解決臨床上常見之細菌抗藥性問題，並提供細菌感染的新治療方式。另一方面，目前並無可靠的方法可用以診斷乳羊的非臨床性乳房炎。因此本研究亦建立乳鐵蛋白的酵素連結免疫分析法，用以研究羊乳中的乳鐵蛋白濃度與乳品質或乳房感染的相關性。經檢測正常桶羊乳後發現，羊乳中的乳鐵蛋白濃度與其美藍還原試驗（methylene blue reduction test）所需時間成負相關性；當桶羊乳的美藍試驗時間為大於8小時、介於5至8小時或小於5小時時，其乳中的平均乳鐵蛋白濃度分別為167、218和304 g/ml。此外，美藍還原試驗時間大於8小時之桶羊乳，其乳中的乳鐵蛋白濃度顯著低於試驗時間小於8小時之桶羊乳。另外，由臨床上個別乳和四頭羊的動物實驗可知，臨床性乳房炎和潛在性乳房炎乳中的乳鐵蛋白濃度均顯著高於正常乳中的濃度。而於動物實驗中，當四頭羊的其中一個乳分房接種金黃色葡萄球菌後，乳中乳鐵蛋白濃度可從低於30 g/ml（第1天）迅速升高為1000 μg/ml （第4天）。於第4天後，乳鐵蛋白濃度逐漸下降，並維持100至300 g/ml至第14天。而對照組乳中的乳鐵蛋白濃度於實驗進行期間均低於100 μg/ml。此外，實驗組乳中的乳鐵蛋白濃度於第3天後均顯著高於對照組乳中的濃度。由此可知，乳中的乳鐵蛋白濃度變化與乳品質或乳房之細菌感染有關，所以羊乳中的乳鐵蛋白濃度變化或能作為乳品質之指標。|
Lactoferrin is a natural antibacterial protein in mammals. To deal with the antibiotic-resistant bacteria infection in the fields, the antibacterial activity and the availability of this protein was studied. To design short and potent analogs of bovine lactoferricin, several peptides were synthesized using the concepts of lipophilic bulk and cationic charge. Thus, basic residues of bovine lactoferricin (bLf 20-29; residues 20 to 29) were modified by substitution with arginine or lysine and nonbasic residues were modified by substitution with tryptophan, phenylalanine, or isoleucine. Peptides were evaluated by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis. It was observed that all synthesized peptides possessed different potency of antibacterial activity against selected bacterial strains. Thus, the arginine, lysine, tryptophan and isoleucine residues were all related to the antibacterial potency of antibacterial peptides. Moreover, bovine lactoferricin has been found to retain the antibacterial potency after sterilization by autoclave. Thus, an E. coli expression system was established to express a large quantity of bovine lactoferrin fusion protein. The expressed protein was intended to be sterilized by autoclave and be applied in fields directly. However, this protein and its protease hydrolyse showed no antibacterial activity. Since bovine lactoferrin is easily available, the antibacterial activity of lactoferrin hydrolysate in combination with antibiotics of different antibacterial mechanisms was studied. Partial synergy of growth-inhibitory activity between bLf-lysate with the gentamicin, cephalothin, cefamandole and rifampicin against antibiotic-resistant E. coli strains was found and synergistic bactericidal activity against the antibiotic-resistant E. coli strains when bLf-lysate was combined with rifampicin. These findings could be used to solve the antibiotic-resistant bacteria problem in fields. Presently, there is no reliable method to detect subclinical mastitis in dairy goats. Thus, an enzyme-linked immunosorbent assay (ELISA) was established to detect the lactoferrin concentration (LFC) in goat milk. The relationship between the variation of LFC in milk and milk quality or intramammary infection was investigated. The results indicated that the LFC in bulk milk negatively correlate to the methylene blue reduction test (MBRT) time of milk. Moreover, the mean LFC in bulk milk of more than 8 hours, 5 to 8 hours or below 5 hours of MBRT time was 167, 218 or 304 g/ml, respectively. The mean LFC in milk of more than 8 hours of MBRT time was significantly lower than that of below 8 hours of MBRT time. Otherwise, the LFC in clinical and subclinical mastitic milk was observed to be significantly higher than that in normal milk. In animal model (4 goats), one of the healthy udder halves (totaled four) of each goat was inoculated with Staphylococcus aureus. The daily LFC of the infected milk increased from below 30 μg/ml (day 1) to 1000 μg/ml (day 4) and then gradually decreased to 100 to 300 μg/ml (day 10), where it remained at this level till the end of experiment (14 day). In contrast, daily LFC in control milk were below 100 μg/ml during the experiment. Moreover, the LFC was significantly higher in infected milk than that in normal milk after day 3. Since milk LFC was found to be associated with the milk quality or intramammary infection, it may be used as an index for intramammary infection in goats.
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