摘要
本实验室已经从新疆家蚕克隆获得抗菌肽基因Cecropin-XJ,通过原核系统和真核系统表达获得了抗菌肽,并对抗菌肽的抗菌特性进行了研究。研究表明,新疆家蚕抗菌肽具有很强的热稳定性、能够杀灭氨苄青霉素抗性菌、对酸碱盐、人造胃酸有一定的耐受性,抗菌谱广,能够不同程度地杀灭革兰氏阳性菌和革兰氏阴性菌。虽然已对新疆家蚕抗菌肽的特性进行了研究,但其结构与功能的关系及抗菌机理还没有完全揭示,本研究采用PCR扩增和人工合成基因的方法,对新疆家蚕抗菌肽基因进行修饰,进行体内抑菌实验与原核表达蛋白的抑菌活性研究,探讨其结构与活性的关系,同时研究了新疆家蚕抗菌肽对南宁麻花雏鸡促进生长及对血清激素水平、生理生化指标的影响:利用紫外光谱及以溴化乙锭为荧光探针的荧光光谱方法研究抗茵肽Cecropin-XJ与金黄色葡萄球菌(Staphylococcus aureus)DNA在体外的相互作用,通过紫外分光光度法研究抗菌肽的抑菌动力学,并采用扫描电镜和透射电镜观察抗菌肽作用于金黄色葡萄球菌后的超微结构;通过核酸免疫法制备了鼠抗新疆家蚕抗菌肽的抗体,结合免疫胶体金技术和透射电镜技术观察抗菌肽在细菌体内的定位情况,探讨新疆家蚕抗菌肽的作用机理。主要研究结果如下:
(1)抗菌肽的α-螺旋、两亲性、疏水性、净正电荷数、关键氨基酸的替换等参数是相互依赖、相互影响,协同发挥作用,任何一个参数的改变都会影响抗菌肽整体的活性。α-螺旋是抗菌肽功能有效性的结构基础,但其所处的位置并不影响抗菌活性;两亲性结构,是抗菌肽与生物膜相互作用的重要结构;疏水性程度必须保持在一定的范围内;在一定范围内增加抗菌肽的阳离子能够增加抗菌活性,但正电荷数和抗菌活性之间无绝对正相关性;色氨酸的存在及抗菌肽的C-末端酰胺化有利于增强抗菌肽的抗菌活性。
(2)与对照组相比,1.5、3.5 mL/L添加抗菌肽的试验组雏鸡增重效果显著(P＜0.05),试验组血清总蛋白浓度差异不显著(P＞0.05),尿素氮水平下降显著(P＜0.05),血清胰岛素样生长因子-1(IGF-1)水平显著升高(P＜0.05)。在28日龄,试验组的三碘甲状腺原氨酸(T3)浓度高于对照组,差异不显著(P＞0.05),甲状腺素(T4)水平下降显著(P＜0.05);在56日龄,试验组的T3水平显著降低(P＜0.05),但T4水平高于对照组,差异显著(P＜0.05)。
(3)真核表达载体pcDNA3/Cecropin-XJ和peDNA3/Ceeropin-XJ4R在小鼠肝脏内能获得有效表达。所制备的鼠抗新疆家蚕抗菌肽的抗血清具有较高的免疫反应性和特异性;实验组血清抗体水平显著高于对照组(P＜0.05),pcDNA3/Cecropin-XJ4R组的抗体水平高于pcDNA3/Cecropin-XJ组,但差异不显著。
(4)利用紫外光谱及以溴化乙锭为荧光探针的荧光光谱方法研究抗菌肽Cecropin-XJ与金黄色葡萄球菌DNA在体外的相互作用,计算获得抗菌肽与DNA的结合常数和成键位点数。结果显示,抗菌肽使DNA发生了明显的增色效应,并使DNA的荧光强度增强,抗菌肽能与EB竞争性的结合DNA,表明抗菌肽可能与DNA双螺旋的沟槽结合;在抗菌肽存在下,DNA与EB作用的结合常数和成键位点数都发生变化,表明抗菌肽以嵌入和非嵌入两种方式与DNA相互作用。
(5)通过紫外分光光度法研究抗菌肽的抑菌动力学,并采用扫描电镜和透射电镜观察抗菌肽作用于金黄色葡萄球菌后的超微结构,对抗菌肽作用机理进行初步探讨。结果表明,抗菌肽抑菌作用比较明显,抗菌肽的活性与作用时间有关。抗菌肽可能是通过“桶-板”模式渗透细胞膜,从而影响细胞膜的结构和功能,使细胞膜形成许多孔道,增强了金黄色葡萄球菌细胞的通透性,造成细胞内的原生质扩散,并从孔道向胞外渗漏,影响了细菌的代谢系统,从而起到抑菌、杀菌作用。抗菌肽使金黄色葡萄球菌细胞内容物大量渗漏而死亡,死亡细胞的细胞壁保持完整,表明细胞膜是抗菌肽作用的主要靶位点。
(6)新疆家蚕抗菌肽处理金黄色葡萄球菌不同时间后,分别用抗菌肽抗体和胶体金标记的二抗处理,透射电镜观察的结果表明,抗菌肽的抗菌作用与浓度和时间相关,并定位于细胞膜和细胞质中。
这些研究结果为进一步改造新疆家蚕抗菌肽,获得抗菌活性更高的抗菌肽,提供了科学依据,通过对Cecropin-XJ作用于S.aureus的机理探讨,提出了Cecropin-XJ以渗透细胞膜和与DNA结合两种作用机理抑杀金黄色葡萄球菌,对于丰富和理解cecropins的作用机制做出了一定的贡献。本研究为研发抗菌肽成为新一代替代抗生素的抗菌药物,尤其是针对抗生素抗性菌的药物提供了依据,为新疆家蚕抗菌肽在新疆畜牧业生产中的应用奠定了理论基础。
Cecropin-XJ gene was isolated from Xinjing silkworm and the corresponding full peptide has been expressed in Pichia yeast and E.coli in our laboratraty.Kinetic studies have shown that the Cecropin-XJ has an effective antibacterial activity.It has exhibited an extreme heat-stable property and the ability to kill ampicillin-resistant S.aureus.Moreover,It has been observed that the Cecropin-XJ was highly tolerant to extreme acidic,basic,and high salt environments as well as resistant to 24-hour digestion by artificial gastric juice.With a broad spectrum of antibacterial activities,the Cecropin-XJ is able to inhibit both Gram positive and Gram negative bacteria.
Although it has been biologically characterized,the structure-function relationship and antibacterial mechanisms of the Cecropin-XJ have not yet been clarified.It has not known whether Cecropin-XJ antibacterial mode of action is due to their effects on membrane,or their effects on intracellular targets such as DNA or the both.In this study,there are five Cecropin-XJ genes were modified by PCR and three other new genes were synthesized. Growth curves of the transformant E.coli containing different new Cecropin-XJ genes were performed to determine the antibacterial activities.To further characterize the antibacterial activity of modified cecropin-XJ,the bacterial inhibition experiment in vitro was also performed.At the same time,the effects of Cecropin-XJ from Xinjiang silkworm on growth-enhancing,serum hormone levels and physical and chemical index in chicken were performed.Mouse antibodies against Cecropin-XJ were generated by DNA immunization. The interaction of the antimicrobial peptide Cecropin-XJ from Xinjiang silkworm and Staphylococcus aureus DNA was investigated by ultraviolet spectra and fluorescence spectra in vitro.The interaction mode was studied by using ethidium bromide(EB) as an extrinsic fluorescence probe.Ultrastructural observation of S.aureus treated with Cecropin-XJ on Scanning Electron Microscope and Transmission Electron Microscope were performed.The localization of immuno-labelled antimicrobial peptides was studied using Transmission Electron Microscope.The main results are as follows:
(1) It was showed that structural parameters includingα-helix,amphipathicity, hydrophobicity,net positive charge,crucial amino acid substitution and so on are interdependent,and therefore,modification of one parameter often leads to significant changes to one or more of the others.Alpha-helix plays an important role in the function of antibacteria,the location of which was of no importance to cecropin-XJ.Amphipathicity may play a more predominant role regarding interaction with target membranes.Hydrophobicity is an essential feature for interactions between antimicrobial peptide and membrane,but its degree must be kept in a certain range.Within the certain range,increasing peptide cationicity is generally associated with increasing antimicrobial potency.However,there is a limit beyond which increasing positive charge no longer confers increased activity.Tryptophan and C-terminal acylation play a crucial role in killing bacteria for Cecropin-XJ.
(2) The results showed that the absolute weight of chicken reared by tap water supplemented with 1.5,3.5 mL/L Cecropin-XJ increased significantly than control group(P＜0.05).Compared with the control group,there was no significant difference in serum total protein of experimental groups(P＞0.05),but the concentration of serum urea nitrogen decreased significantly in experimental groups supplemented with 1.5,3.5 mL/L Cecropin-XJ (P＜0.05).Serum IGF-1 levels of experimental groups increased significantly than control group.Experimental groups' serum T3 levels at 28 days were higher than those of control group,but there was no significant difference.Serum T4 levels of experimental groups were significantly lower than those of control group at 28 days(P＜0.05).T3 levels of experimental groups were significantly lower than those of control group chickens at 56 days (P＜0.05),but T4 levels of experimental groups were significantly higher than those of control group chickens.
(3) It was showed that the recombinant plasmids were successfully expressed in mouse liver and induced antibody with higher immune reactivity and specificity in mice.The specific antibody levels of mice immunized with the recombinant plasmid were significantly higher than those with empty vector(P＜0.05).Antibody levels of mice immunized with pcDNA3-Cecropin-XJ4R were higher than those of pcDNA3-Cecropin-XJ,but there was no significantly difference.
(4) The increase in absorbance of DNA samples at 260nm due to the addition of Cecropin-XJ was measured.It is called hyperchromicity of DNA,which can provide a direct measure of the degree of base-pair unstacking.The unstack results in the loss of duplex helix and then leads to the duplex helix relaxing.At the same time,the interaction mode was studied by using ethidium bromide(EB) as an extrinsic fluorescence probe.With the addition of Cecropin-XJ,the intensity of intrinsic fluorescence absorbance of DNA at 593nm increases greatly.It is suggested that the addition of Cecropin-XJ may stack with the base-pair of DNA or expose the aromatic amino acides,which lead to the enhancing of DNA fluorescence intensity.Subsequently the competition between Cecropin-XJ and EB to combine with DNA was found.It suggested that the style was groove binding and intercalation in the interaction between Cecropin-XJ and double helix DNA.Furthermore,in this study,the binding constant and binding number of Cecropin-XJ complex with DNA was determined.The binding constant and number of EB complex with DNA changed with addition of Cecropin-XJ.It showed that the interaction between Cecropin-XJ and DNA was based on intercalation and non-intercalation.Taken together,it indicated that Cecropin-XJ can bond to S.aureus chromosome DNA,which suggests existing DNA synthesis inhibition mechanism. Meanwhile,these results contribute to explain the molecular mechanism of antimicrobial peptide from the interaction style and structural charateristic of Cecropin-XJ and S.aureus DNA.
(5) It was showed that the ability of cecropin-XJ to kill S.aureus is effective and time-dependent.It may be the "barrel-stave pore" mechanism that cecropin-XJ quickly penetrated and damaged the membrane.And then it was observed that cecropin-XJ make effects on the cytoplasmic membrane,disturbing the ordered arrangement of membrane lipid and changing the permeability of cytoplasmic membrane with the cytoplasmic contents of the cells leaking out.Finally,the affected S.aureus was died due to leakage of the cell contents.
The intact cell wall was left,not losing their shape after treated with cecropin-XJ,which strongly showed that no cell wall disintegrated,althought cecropin-XJ acted on S.aureus and led to cell death.These results indicated cytoplasmic membrane is the main target attacked by cecropin-XJ.
(6) The immunolabelled localization of antimicrobial peptides was studied using transmission electron microscopy.Staphylococcus aureus was exposed to Cecropin-XJ. Cecropin-XJ was found in the cytoplasm and membrane of bacteria.Cecropin-XJ could effectively kill Staphylococcus aureus in the dosage-and time-dependent way.
Based on these results,modification of Ceeropin-XJ to improve antimicrobial activity may be important in the rational design of novel therapeutic agents.To our knowledge,this study represents the first report on two different mechanism of action of Cecropin-XJ to kill S. aureus.First,membrane mechanism of Cecropin-XJ was emphasized by the SEM and immuno-TEM.Second,Cecropin-XJ may interact with DNA to inhibit the DNA replication and transcription,and then inhibit S.aureus growth and development.In addition to increasing our understanding of a novel mechanism of action of Cecropin-XJ,the study presents a potential antibiotic to overcome S.aureus,especially in killing antibiotic-resistant S.aureus, and contributes to develop a novel antibiotic for future therapeutic purposes.These findings could lead to a broad application prospect in the medical industry and domestic animal industry of Xinjiang.
引文
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