泰山松花粉多糖的成分分析及其对B亚群禽白血病的免疫调理作用

发布时间：2018-04-25 01:38

本文选题：松花粉多糖 + B亚型禽白血病病毒　；参考：《山东农业大学》2015年博士论文

【摘要】：禽白血病(Avian Leucosis)是由禽白血病病毒(Avian Leukosis Virus,ALV)和禽肉瘤病毒(Avian Sarcoma Virus,ASV)群中的病毒感染引起的多种肿瘤性疾病的统称。该病在蛋鸡、肉鸡以及我国地方品系鸡群中都有发生,造成鸡群生产性能降低、免疫抑制、肿瘤性死亡等危害,尤其是ALV感染引起的亚临床感染,导致感染鸡生产性能低下和免疫抑制,从而引起其它病毒和细菌的混合感染或继发感染,导致继发感染或共感染,给养禽业造成重大经济损失。对于禽白血病的防制,目前尚缺乏有效的药物和疫苗,只能通过种群净化来实现对该病的控制。在过去的几十年中,由于未能采取有效地净化措施,禽白血病在养禽地区广泛的流行和散播,致使对其防控更加耗时耗力。因此,亟需探索新的方法控制ALV的散播、减少感染ALV感染的亚临床症状,减少该病带来的危害。目前,多种植物多糖作为新型的添加剂被证明有抗病毒、抗肿瘤、抗氧化、抗辐射等多种生物学作用。本实验室之前的研究发现泰山松花粉多糖(Taishan pine pollen polysaccharose,TPPPS)能显著增强奇异变形杆菌和禽波氏杆菌亚单位疫苗的免疫效果,促使更强的免疫应答反应;提高獭兔的生产性能;对免疫抑制小鼠有免疫恢复作用。然而,前面所用TPPPS为水提醇沉法所提的总多糖,至今仍不清楚TPPPS中的有效活性成分及相应的生物学特性,因此限制了人们对TPPPS的认识及应用。基于以上考虑,本研究首先分析了TPPPS的成分和理化特性,并鉴定了每种成分的生物学活性;进一步对不同组份抑制B亚群禽白血病病毒(ALV-B)增殖的特性进行了分析;最终,通过建立感染感染ALV-B的鸡免疫抑制模型,探究了TPPPS对ALV-B诱导的免疫抑制的免疫调理和恢复作用,为探索研发防制禽白血病的新途径奠定基础。本研究分以下三部分内容:1.TPPPS部分理化特性和生物学活性的分析1.1 TPPPS的柱层析纯化及各组分分子量和单糖组成的检测为进一步研究TPPPS的功能和发挥作用的机理,本研究将粗提的多糖用DEAE纤维素柱和Sephadex G-200凝胶柱进一步的纯化。结果显示,松花粉多糖主要包括三种组分(分别命名为TPPPS1、TPPPS2和TPPPS3),并且分离得到了这三种组分的均一多糖。利用高效凝胶渗透色谱法(HPGPC)和高效液相色谱(HPLC)检测三种多糖组分的分子量和单糖组成。结果表明TPPPS1、TPPPS2和TPPPS3的分子量分别为56、25和128 k Da。TPPPS由甘露糖、核糖、木糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖等构成,TPPPS1-3组分之间各单糖组成的种类和比例存在明显不同。以上研究结果提示这三种组份可能具有不同的生物学特性。1.2 TPPPS体外抗氧化性、免疫增强活性、抗病毒活性的检测TPPPS各组分的分子量和单糖组成差异显著,表明TPPPS1-3的生物学活性可能存在明显的差异。为了进一步了解TPPPS的生物活性,本研究对TPPPS、TPPPS1、TPPPS2和TPPPS3的三种体外生物活性进行了检测。结果表明,TPPPS2和TPPPS3显著促进脾淋巴细胞的增殖,并且TPPPS3的效果强于TPPPS2;TPPPS3增强细胞因子白介素2(IL-2)和肿瘤坏死因子(TNF)的分泌,而TPPPS2仅可以显著提高IL-2的分泌;值得注意的是,TPPPS1没有以上作用,但诱导NO产生的效果最为显著,表明TPPPS1具有最显著的抗氧化活性;50μg/m L TPPPS3对ALV-B增殖作用最为显著,并且作用的主要时期是在病毒的吸附期。本研究为使用TPPPS1-3合理比例的进一步研究可促进有效的免疫调节剂的开发和分析TPPPS的体外的抗病毒机制奠定了基础。2.TPPPS体外抗病毒活性机制的分析2.1阻断ELISA方法的建立为研究TPPPS对ALV-B增殖的显著抑制主要发生在病毒吸附宿主细胞的过程中的机制,本研究根据ALV-B的吸附过程中起重要作用的两个蛋白,病毒的囊膜糖蛋白gp85和宿主的受体蛋白tv-b,建立两个ELISA方法。用原核表达的方法表达出gp85和tv-b蛋白,并制备这两个蛋白的多克隆抗体,用这两个蛋白和抗体建立两个受体-病毒ELISA方法(方法I:gp85—tv-b—tv-b抗体—酶标二抗;方法II:tv-b—gp85—gp85抗体—酶标二抗)。用方阵试验确定ELISA方法的各个条件,结果显示方法I中gp85,tv-b,tv-b抗体的浓度,阴阳性临界值分别是16μg/m L,32μg/m L,1:80,0.178,方法II中tv-b,gp85,gp85抗体的浓度分别是32μg/m L,32μg/m L,1:80,0.194,封闭液和TMB显色时间都是5%脱脂奶粉和15 min。本试验成功建立两个ELISA方法用于分析TPPPS体外抗病毒活性机制。2.2 TPPPS体外抗病毒活性机制的分析利用建立的两个受体-病毒ELISA方法,在gp85蛋白(或tv-b蛋白)包被ELISA板之后加入200μL不同浓度(100,101,102,103,104,105μg/m L)的TPPPS3溶液孵育,进行后续试验观察结果的变化。结果显示,TPPPS3可以显著降低ELISA方法I中的P/N值,对方法II的影响不显著,这表明多糖能与病毒的gp85蛋白的结合,并且不能与tv-b受体蛋白结合,这种结合干扰了病毒与tv-b受体蛋白的结合,这是TPPPS抑制了病毒扩增的机制之一。本研究为进一步认识TPPPS在ALV-B吸附宿主细胞过程中的作用,为研究TPPPS抑制ALV-B在雏鸡体内增殖的机制奠定基础,为TPPPS在禽白血病防制方面的应用提供了依据。3.TPPPS对感染ALV-B雏鸡免疫抑制模型的调理作用3.1感染ALV-B雏鸡免疫抑制模型的建立鸡群感染ALV后可造成1-2%的死亡率(偶尔可达20%或更高),而大部分鸡只有亚临床感染,主要是表现是生产性能下降和免疫抑制。本研究选用50只1日龄雏鸡感染ALV-B,分别在感染后1-9周监测感染鸡的各项生理指标的变化,建立免疫抑制模型,检测指标包括:发病率与死亡率,体重与免疫器官指数,抗ALV-B特异性抗体水平,γ干扰素(IFN-γ)与白介素2(IL-2)水平,T淋巴细胞增殖率,CD4+和CD8+淋巴细胞亚群比例等。结果显示通过对以上指标的检测未发现感染鸡死亡,但其生长状况显著差于正常鸡,并且免疫器官发育迟缓,免疫反应不明显,淋巴细胞活性降低,CD4+和CD4+/CD8+显著下降等。结果表明,感染鸡的免疫系统受到显著地免疫抑制,免疫抑制模型建立成功,为分析感染ALV-B雏鸡的免疫反应变化提供了依据。3.2 TPPPS对免疫抑制模型雏鸡的调理作用据报道TPPPS对环磷酰胺导致的免疫抑制小鼠有免疫恢复作用,本研究通过给感染ALV-B的免疫抑制模型雏鸡颈部皮下注射TPPPS,分析多糖对免疫抑制鸡的免疫调理作用,同时在雏鸡一周龄后免疫新城疫病毒(NDV)疫苗,之后检测各项免疫指标,用以分析TPPPS对感染ALV-B雏鸡免疫抑制模型的调理恢复作用。研究结果显示,TPPPS组雏鸡的体重和免疫器官指数,外周血淋巴细胞增殖率,CD4+淋巴细胞亚群和CD4+/CD8+比值,IL-2和IFN-γ的分泌,和ALV-B的抗体阳性率都显著增加,并且剂量为400 mg/kg(每千克鸡)的TPPPS的效果是最显著的。此外,在400 mg/kg TPPPS组抗新城疫病毒的抗体滴度也显著高于其它试验组。这些结果表明,TPPPS可以用作一种免疫增强剂,以减轻ALV-B造成的免疫抑制现象,为防制禽白血病提供新的思路和途径。
[Abstract]:Avian leukosis (Avian Leucosis) is a general name for a variety of tumor diseases caused by the virus infection of avian leukemic virus (Avian Leukosis Virus, ALV) and avian sarcoma virus (Avian Sarcoma Virus, ASV). This disease occurs in laying hens, broilers and local chicken flocks in our country, resulting in reduced production performance, immunosuppression and swelling of the chickens. The risk of tumor death, especially the subclinical infection caused by ALV infection, leads to low production performance and immunosuppression of infected chickens, resulting in mixed infection or secondary infection of other viruses and bacteria, resulting in secondary infection or co infection, causing major economic losses in poultry industry. Drugs and vaccines can only be controlled by population purification. In the past few decades, due to failure to take effective purification measures, avian leukosis has been widely spread and spread in poultry areas, resulting in more time consuming and power consumption. Therefore, it is urgent to explore new ways to control the spread of ALV and reduce the infection of ALV. Clinical symptoms, reducing the harm caused by the disease. At present, a variety of plant polysaccharide as a new additive has been proved to have antiviral, anti-tumor, antioxidant, anti radiation and other biological effects. Previous studies in this laboratory found that Taishan pine pollen polysaccharide (Taishan pine pollen polysaccharose, TPPPS) can significantly enhance the strain of Proteus mirabilis and The immune effect of the subunit vaccine of Pasteurella fowl promotes the stronger immune response, improves the production performance of the Rex Rabbit and has the immune recovery effect on the immunosuppressed mice. However, the previous TPPPS is the total polysaccharide extracted by the water extraction and alcohol precipitation method, which is still not clear to the active components in the TPPPS and the corresponding biological characteristics. Therefore, the restriction is limited. Based on the above considerations, this study first analyzed the composition and physicochemical properties of TPPPS, and identified the biological activity of each component, and further analyzed the characteristics of the inhibition of the proliferation of B subgroup of avian leukosis virus (ALV-B) by different components, and finally, the immunosuppression of chickens infected with ALV-B was established. The model, exploring the immunomodulation and recovery effect of TPPPS on ALV-B induced immunosuppression, lays the foundation for exploring new ways to develop anti avian leukemia. The following three parts: analysis of the physical and chemical properties and biological activities of 1.TPPPS, analysis of 1.1 TPPPS column chromatography and the detection of molecular weight and monosaccharide composition of each component In order to further study the function and mechanism of TPPPS, the crude polysaccharide was further purified by DEAE cellulose column and Sephadex G-200 gel column. The results showed that the polysaccharide mainly consisted of three components (named TPPPS1, TPPPS2 and TPPPS3 respectively), and the polysaccharides of these three components were obtained. The molecular weight and monosaccharide composition of three polysaccharides were detected by high performance gel permeation chromatography (HPGPC) and high performance liquid chromatography (HPLC). The results showed that the molecular weights of TPPPS1, TPPPS2 and TPPPS3 were 56,25 and 128 K Da.TPPPS, respectively, consisting of mannose, ribose, xylose, glucuronic acid, galacononic acid, glucose, galactose and Arabia sugar, etc. The species and proportion of monosaccharides among the -3 components were distinctly different. The above results suggest that these three components may have different biological characteristics,.1.2 TPPPS in vitro antioxidant, immune enhancement, and antiviral activity, the molecular weight of TPPPS components and monosaccharide components are significantly different, indicating the biological activity of TPPPS1-3. There may be obvious differences. In order to further understand the bioactivity of TPPPS, three in vitro biological activities of TPPPS, TPPPS1, TPPPS2 and TPPPS3 were detected. The results showed that TPPPS2 and TPPPS3 significantly promoted the proliferation of spleen lymphocytes, and the effect of TPPPS3 was stronger than TPPPS2; TPPPS3 enhanced cytokine interleukins 2 (IL-2) and swelling. The secretion of tumor necrosis factor (TNF), and TPPPS2 only can significantly increase the secretion of IL-2; it is worth noting that TPPPS1 has no above effect, but the effect of induced NO is the most significant, indicating that TPPPS1 has the most significant antioxidant activity; 50 mu g/m L TPPPS3 is the most significant proliferation of ALV-B, and the main period of action is the virus sucking. Further study on the use of a reasonable proportion of TPPPS1-3 to promote the development and analysis of effective immunomodulators for the development and analysis of the anti-virus mechanism of TPPPS in vitro; analysis of the mechanism of the antiviral activity of.2.TPPPS in vitro: 2.1 the establishment of the ELISA method of blocking the proliferation of TPPPS mainly occurs in the virus. In the process of adsorption of host cells, this study establishes two ELISA methods based on the two proteins that play important roles in the adsorption process of ALV-B, the viral capsule glycoprotein gp85 and the host receptor protein tv-b. The gp85 and tv-b proteins are expressed by the prokaryotic expression method, and the polyclonal antibody of these two proteins is prepared, and the two eggs are used. White and antibodies establish two receptor virus ELISA methods (Methods I:gp85 - tv-b - tv-b antibody - enzyme labeled two, II:tv-b - gp85 - gp85 antibody - enzyme standard two). Using square array test to determine the conditions of ELISA method, the results show that the concentration of gp85, tv-b, tv-b antibodies in I, and the critical value of yin and yang are 16 mu, 32 micron, respectively, 0.178, the concentration of tv-b, gp85, gp85 antibody in method II is 32 g/m L, 32 mu g/m L, 1:80,0.194, blocking solution and TMB color time are all 5% degreased milk powder and 15 min. experiment, and two ELISA methods are successfully established to analyze the mechanism of antiviral activity in vitro The virus ELISA method, after the gp85 protein (or tv-b protein) package was incubated with the TPPPS3 solution of 200 mu L with different concentrations (100101102103104105 g/m L), was incubated after the ELISA plate. The results showed that TPPPS3 could significantly reduce the P/N value in ELISA I, which showed that the effect of polysaccharide was not significant. The binding of the gp85 protein of the virus and not binding with the tv-b receptor protein, which interferes with the binding of the virus to the tv-b receptor protein, is one of the mechanisms that TPPPS inhibits the amplification of the virus. This study is to further understand the role of TPPPS in the process of ALV-B adsorption on the host cells and to study the inhibition of the proliferation of ALV-B in chicks by TPPPS. The mechanism lays the foundation for the application of TPPPS in the prevention and control of avian leukaemia, which is based on.3.TPPPS's regulating effect on the Immunosuppressive Model of infected ALV-B chicks. 3.1 infected ALV-B chicken immune inhibition models can cause the mortality of 1-2% after chicken group infection ALV (occasionally up to 20% or higher), and most chickens only have subclinical infection. In this study, 50 1 day old chickens were infected with ALV-B, and the changes of the physiological indexes of infected chickens were monitored at 1-9 weeks after infection, and the immunosuppressive model was established. The detection indexes included the incidence and mortality, the body weight and the immune organ index, the anti ALV-B specific antibody level, and interferon gamma (IF). N- gamma and interleukin 2 (IL-2) level, T lymphocyte proliferation rate, CD4+ and CD8+ lymphocyte subgroup ratio. The results showed that no infected chickens died, but the growth status of the chickens was significantly worse than that of normal chickens, and the immune organs were retarded, the immune response was not obvious, the lymphocyte activity decreased, and CD4+ and CD4+/CD8+ showed. The results showed that the immune system of infected chickens was significantly immunosuppressed and the immunosuppressive model was established successfully. In order to analyze the changes of immune response of infected ALV-B chickens, the regulation of.3.2 TPPPS on the Immunosuppressive Model chicks was reported to have been reported by TPPPS to the immunosuppressive mice induced by cyclophosphamide. In this study, the immunomodulation effect of polysaccharides on immunosuppressed chickens was analyzed by subcutaneous injection of TPPPS in the neck of chickens infected with the Immunosuppressive Model of ALV-B. At the same time, the immunization of Newcastle disease virus (NDV) after one week of the chicks was immunized, and then the immune indexes were detected to analyze the adjustment and recovery of the Immunosuppressive Model of ALV-B chicks infected with TPPPS. The results showed that the body weight and immune organ index, the proliferation rate of peripheral blood lymphocyte, the ratio of CD4+ lymphocyte subsets and CD4+/CD8+, the secretion of IL-2 and IFN- gamma, and the positive rate of antibody to ALV-B were significantly increased in the TPPPS group, and the effect of TPPPS of 400 mg/kg (per kilogram chicken) was the most significant. In addition, at 400 mg/kg The antibody titer of anti Newcastle disease virus in group TPPPS is also significantly higher than that of other experimental groups. These results suggest that TPPPS can be used as an immune enhancer to alleviate the immunosuppression caused by ALV-B and provide new ideas and ways for preventing avian leukemia.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S853.7

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