【作者】 单媛媛；

【导师】 马美湖；

【作者基本信息】 华中农业大学 ， 食品科学， 2013， 博士

【摘要】 粘蛋白是生物机体内最古老的防御体系的重要成分之一,在众多生命活动过程中发挥着极其重要的作用。然而几乎所有粘蛋白目前已知的功能均是在哺乳动物中发现的,对禽蛋中粘蛋白的研究相对较少。本文以鸡蛋蛋清中的粘蛋白(ovomucin)为研究对象,以ovomucin的结构与功能为核心,在纯化出高纯度和高活性蛋白质的基础上,研究了提高ovomucin溶解性的方法,并对其体外抗菌、抗病毒感染的活性与生化机制开展了系列研究工作。主要研究内容和结果如下：首先采用分步等电点盐析结合凝胶过滤色谱法从鸡蛋清中分离纯化ovomucin,其过程为：50mmol/L CaCl2溶液制备较高含量的ovomucin,后用50mmol/L MgCl2进一步除去溶菌酶,再经Sephacryl S-300HR凝胶过滤法纯化,进样体积为2mL,洗脱液为含0.05mol/L MgCl2的Tris-HCl (20mmol/L pH8.4),流速为0.5mL/min。质谱鉴定结果表明所得蛋白质为ovomucin,不含其它蛋白质肽段信息。HPLC和SDS-PAGE分析结果显示其纯度为99.13%。采用此方法制备的ovomucin终产率为(57.03±1.22)%,总回收率为(40.87±0.76)%。纯化后的ovomucin对新城疫病毒(NDV)LaSota株的半数抑制浓度(IC50)为5.78±0.09μg/mL.酶联免疫吸附实验(ELISA)结果表明低浓度MgCl2可以显著提高ovomucin对NDV的粘附能力(P<0.05)。氨基酸分析显示获得的ovomucin富含Ala, Thr, Pro和Val,具有典型的粘蛋白特性,同时含有较多硫酸基、唾液酸、糖醛酸和己糖,具有作为抗菌抗病毒研究的潜在结构基础。研究了提高ovomucin溶解性的方法与机制。溶解性测定结果表明,水不溶性ovomucin在1mol/L精氨酸中溶解度为(21.14±1.47)%,是其在蒸馏水(1.96%)中的10倍以上。荧光猝灭实验表明精氨酸在低浓度时通过静电作用与ovomucin形成静态复合物,二者在不同温度下结合常数Ka分别为3.282L·mol-1(298K)、2.404L·mol-1(308K)和1.904L·mol-1(313K),结合位点数n分别为1.141(298K)、1.130(308K)和1.118(313K)。电导法和芘荧光探针实验结果发现,精氨酸浓度小30mmol/L时主要通过静电作用与ovomucin结合,未引起ovomucin结构的显著变化；精氨酸浓度在30-100mmol/L时,复合体中出现大量疏水微区。ANS和FT-IR分析结果表明,在精氨酸浓度大于100mmol/L以后,ovomuicn二级结构中的无规则卷曲增加,折叠结构及螺旋结构减少,分子间聚集程度减弱,ovomucin的表面疏水性显著降低。粘附试验结果表明,添加100mmol/L精氨酸并未导致ovomucin对NDV粘附活性的显著降低(P>0.05),因此可以应用精氨酸实现ovomucin的非变性有效增溶。测定了ovomucin的抗菌活性及其对环境条件的稳定性。结果表明：ovomucin对大肠杆菌和沙门氏菌最小抑菌浓度分别为0.05mg/mL和0.4mg/mL,相应的抑菌率分别为(61.23±6.5)%和(34.34±4.4)%,而对金黄色葡萄球菌则无明显抑制作用。细菌生长曲线的测定结果发现,0.05mg/mL的ovomucin可以有效抑制大肠杆菌的增殖,但其仅能延缓沙门氏菌的对数生长期。粘附试验结果表明,ovomucin对三种细菌均具有一定的粘附能力,ovomucin浓度由2.5μg/mL增大到30μg/mL时,其对大肠杆菌的粘附率由19.3%升高到64.4%,对沙门氏菌的粘附率由22.1%升高到68.3%,对金黄色葡萄球菌的粘附率由16.3%升高到68.5%,但相同粘度的ovomucin对不同细菌粘附效率之间无显著性差异(P>0.05)。不同pH处理对ovomucin抗菌活性影响显著(P<0.05),在酸性条件下ovomucin对大肠杆菌的抑制率较低,随着pH升高抑菌率增大,在pH7-9条件下对大肠杆菌的抑制效果最好。Ovomucin的抗菌活性对温度具有较好的耐受性,即使经100℃处理10min,ovomucin对大肠杆菌的抑菌率仍可达60%以上。在建立ovomucin抗病毒模型的基础上,通过细胞病变程度、细胞存活率、病毒抑制率以及血凝抑制等指标,评价ovomucin对流感病毒H5N1、H1N1以及新城疫病毒LaSota、Ⅴ-4和Ⅳ系的抑制作用,结果发现ovomucin对五株不同毒株均具有一定的抑制效果,与病毒混合后能明显降低病毒感染力,0.25mg/mL的ovomucin对H5N1、H1N1以及新城疫病毒Ⅱ、Ⅴ-4和Ⅳ系的血凝抑制率分别为(72.89±6.02)%、(64.29±7.94)%、(71.15±6.41)%、(60.32±9.16)%和(74.26±7.35)%。细胞实验发现ovomucin对H5N1早期吸附进入细胞的抑制效果最好,0.25mg/mL的ovomucin对H5N1在MDCK细胞上的病毒抑制率为77.85%。不同来源粘蛋白对H5N1的抗病毒活性存在差异,ovomucin的对H5N1的抑制效果高于牛下颌腺粘蛋白,但是略低于猪胃粘蛋白。为进一步探讨ovomucin抗H5N1的生化机制,通过改变加药方式,从体外细胞水平的抗病毒作用环节结合直接显微观察探讨ovomucin体外抗流感病毒的作用环节,结果表明ovomucin抗病毒作用是通过抑制病毒感染细胞早期的吸附或进入过程实现的。血凝实验和神经氨酸酶抑制实验结果证明ovomucin主要通过抑制H5N1表面血凝素(HA)识别细胞的过程达到抑制病毒侵染的目的,同时ovomucin也起到一定的保护细胞作用。ELISA和双偏振极化干涉测量(DPI)结果表明ovomucin可以与HA抗原发生有效结合；DPI实验进一步分析ovomucin与H5N1表面HA的相互作用过程,发现二者以嵌入式的方式发生结合。因此ovomucin抗HsN1感染的机制为：ovomucin与HA通过嵌入式结合,抑制HA对MDCK细胞受体的识别,从而影响H5N1侵染早期对宿主细胞的吸附和侵入,进而抑制病毒增殖,在这个过程中还伴随着ovomucin和宿主细胞表面发生相互作用来保护细胞免受病毒侵染的过程。采用傅里叶变换红外光谱法和二维相关分析技术研究了ovomucin构象转变与温度之间的关系。结果显示,25-95℃升温过程中ovomucin分子二级结构的变化次序依次为：a-螺旋,p-折叠,p-转角,无规卷曲。Ovomucin构象在pH8.4-9.0之间有中间体存在。红外光谱拟合结果表明低浓度Mg2+与ovomucin糖基结合,未造成ovomucin二级结构的显著改变。而高浓度Mg2+会影响ovomucin的肽骨架及其空间构象,使ovomucin的无规则卷曲结构减少,β-转角和p-折叠结构增加。动态光散射结果表明添加0.1倍质量浓度的Mg2+可以使0.5mg/mL的ovomucin的平均粒度由60.23nm下降到51.39nm,说明Mg2+的加入会使ovomucin的分子结构变得更为紧密。pH诱导ovomucin构象与活性的分析结果表明,随着溶液中p-转角含量由20.7%增加到39%,ovomucin对H5N1血凝的抑制率也相应由53.9%提高到73.1%,Mg2+对ovomucin结构与功能的影响结果发现,加入Mg2+以后,ovomucin二级结构中的p-转角含量由19.11%增加到22.96%,相应的对病毒的粘附率可以由30%左右提高到68.9%。上述结果说明ovomucin结构有序性和紧密程度越高,其抗病毒活性越强。更多还原

【Abstract】 As an important component of the most ancient defense system, mucus gel performs a critical function in defending the epithelial tissues against pathogenic and environmental challenges under different physiological and pathological states. However, almost all known functions of mucins are found in mammals, and knowledge of ovomucin in eggs is relatively limited. The present thesis focused on the structure and function of ovomucin. A series of experiments related to ovomucin has been performed, utilizing the techniques for protein separation and purification, protein structure analysis and antivirus evaluation. The main research contents and results are as follows:An improved procedure comprises a precipitation with0.05mol/L CaCl2followed by precipitation with0.05mol/L MgCl2was developed for the isolation of ovomucin. Gel filtration was then used for further purification,2mL sample with concentration of1mg/mL was applied to a gel filtration system on Sephacry1S-300HR column, eluting with0.02mol/L Tris-HCl buffer (pH8.4) containing0.05mol/L Mg Cl2at a flow rate of1.0mL/min. Ovomucin of high purity (99.13%) was obtained in good yield (302.14mg/100g fresh egg white) via an improved two-step precipitation followed by gel filtration chromatography. The IC50of the preparation for LaSota is5.78±0.09μg/mL and the recovery of the whole process was (40.87±0.76)%. Better adhesion property of ovomucin was observed when low concentration of MgCl2was added in the designed ELASA test, whereas the adhesion property of the pure ovomucin without salts to NDV was lower. Amino acid analysis found that it was rich in Alanine, Threonine and Valine residues, which is confirmed to be a typical mucin. High content of sulfuric acid group, sialic acid, uronic acid and hexose was also detected in the preparation.We demonstrate here that addition of L-arginine at1M can dramatically increase ovomucin solubility by up to10folds. Analysis of fluorescence spectra revealed that arginine and ovomucin formed a static complex via electrostatic interactions. The binding constants (Ka) and number (n) at different temperatures were as follows:Ka=3.282L·mol-1, n=1.141(298K); Ka=2.404L·mol-1, n=1.130(308K), Ka=1.904L·mol-1, n=1.118(313K). The conductance and fluorescence measurements employing pyrene as a probe suggest that the structure of ovomucin does not change measurably if small amounts of arginine are added ([arginine]<30mM). As the arginine concentration is increased (30<[arginine]<100mM), ovomucin swells and more arginine is incorporated in the ovomucin-arginine complexes, leading hydrophobic domains formed. The structural investigation by FI-IR and ANS probe indicated an increase in the proportion of random proportion, accompanied by a decrease in the alpha-helix and beta structures when the arginine concentration increased to above100mM. Additionally, such structural perturbations had no adverse effect on the adhesion activity of ovomucin to NDV.Ovomucin showed obvious anti-bacterial activity to Escherichia coli and Salmonella, with MIC of0.05mg/mL and0.4mg/mL, respectively, while it had no significant effect on the growth of Staphylococcus aureus. The inhibition rate of ovomucin with0.05mg/mL and0.4mg/mL against Escherichia coli and Salmonella was (61.23±6.5)%and (34.34±4.4)%, respectively. E. coli was significantly inhibited during the whole process of the growth cycle after the addition of0.05mg/mL ovomucin. In contrast, ovomucin showed inhibition activity against Salmonella by retarding the logarithmic growth phase. The adhesion rate to E. coli increased from19.3%to64.4%, when the ovomucin concentration increased from2.5(μg/mL to30μg/mL. And adhesion rate to Salmonella increased from22.1%to68.3%, while adhesion rate to Staphylococcus aureus increased16.3%to68.5%, when the same ovomucin concentration change occurred. However, there is no significant difference in adhesion efficiency between different bacterial for the same ovomucin concentration (P>0.05). Better antibacterial effect to E. coli occurred in neutral and alkaline conditions. A thermal stability of antibacterial activity against E. coli can be seen below100℃. The inhibition rate of ovomucin to E. coli is still more than60%even treated at100℃for10min.Antiviral effects against NDV virus (Ⅱ, V-4and Ⅳ strain) and two types of influenza virus were determined. Cytopathic degree of cell, survival rate, and virus inhibition rate and hemagglutination inhibition were used as evaluation index. The anti-viral activity of different mucins was compared as well. It was found that ovomucin had inhibition effect on all of the five tested virus strains. Virus infectivity of the virus can be significantly reduced after be mixed with ovomucin. The hemagglutination inhibition rate of ovomucin with0.25mg/mL against H5N1H1N1, NDV of Ⅱ, V-4and Ⅳ strain was (72.89±6.02)%,(64.29±7.94)%,(71.15±6.41)%,(60.32±9.16)%and (74.26±7.35)%, respectively. Results from cell experiments found that better antivirus activity of ovomucin against H5N1occurred in the early adsorption stage to MDCK. The inhibition rate of ovomucin (0.25mg/mL) to H5N1virus in MDCK cells was77.85%. The inhibitory effect of ovomucin to H5N1is higher than that of bovine submaxillary gland mucin, but slightly lower than that of the porcine stomach mucin. We designed an assay to investigate the time course effect of the virus infection at different concentration of ovomucin. Result revealed that it can inactivate H5N1in vitro by inhibiting the virus from infected cells in early adsorption or entry procedure. Antiviral properties were worth further investigated to identify the active sites, using hemagglutination inhibition test and neuraminidase inhibition test. ELISA and DPI results showed that the ovomucin can interacted effectively with hemagglutinin, the surface antigen of H5N1. DPI result also indicated that ovomucin interacted with hemagglutinin of H5N1by embedded mode, thus resulting in the conformational changes of virus hemagglutinin. Thus, the mechanism that ovomucin strongly inhibited the infection of H5N1on MDCK cells can be interpreted as follows:ovomucin can interact with hemagglutination, the surface protein of the virus, by an embedded manner, thus affect the recognition process of virus to MDCK cell receptor, thereby inhibit the virus infection in the early adsorption and invasion to host cells, achieving the inhibition of virus multiplication. In addition, ovomucin interacts with cell surface to protect cells against viral infection was also accompanied in this process.The order of secondary structural changes in ovomucin as induced by temperature was α-helix,(β-sheet, β-turn, random coil. The existence of intermediate during pH induced unfolding process implied that the conformational transition of ovomucin does not fit the simple two-state mechanism. Mg2+prefers to coordinate with the carbohydrate moiety of ovomucin without significant changes in the secondary structure of ovomucin at a relatively low Mg2+concentration, which affect the microenvironment of aromatic amino acid residues, causing fluorescence quenching of ovomucin. When Mg2+increases to a higher concentration, it will also interact with the protein moiety of ovomucin, giving rise to changes in secondary structure. The DLS results showed that the average particle size of0.5mg/mL ovomucin (60.23nm) increased by the addition of Mg2+to ovomucin (51.39nm). Results from pH-induced the conformation and antiviral activity of ovomucin showed that with the content of beta-turn from20.7%to39%, he hemagglutination inhibition rate of ovomucin against H5N1corresponding increased from53.9%to73.1%. Results from the impact of Mg2+on the structure and function of ovomucin found that, with content of beta-turn from19.11%to22.96%in the secondary structure of ovomucin, the adhesion rate of ovomucin to virus can increased from30%to68.9%. These results indicated that the antivirus activity of ovomucin was increased with the improvement of the ordered and closely structure.更多还原