本文選題：松花粉多糖 + B亞型禽白血病病毒　； 參考：《山東農(nóng)業(yè)大學》2015年博士論文

【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種腫瘤性疾病的統(tǒng)稱。該病在蛋雞、肉雞以及我國地方品系雞群中都有發(fā)生,造成雞群生產(chǎn)性能降低、免疫抑制、腫瘤性死亡等危害,尤其是ALV感染引起的亞臨床感染,導致感染雞生產(chǎn)性能低下和免疫抑制,從而引起其它病毒和細菌的混合感染或繼發(fā)感染,導致繼發(fā)感染或共感染,給養(yǎng)禽業(yè)造成重大經(jīng)濟損失。對于禽白血病的防制,目前尚缺乏有效的藥物和疫苗,只能通過種群凈化來實現(xiàn)對該病的控制。在過去的幾十年中,由于未能采取有效地凈化措施,禽白血病在養(yǎng)禽地區(qū)廣泛的流行和散播,致使對其防控更加耗時耗力。因此,亟需探索新的方法控制ALV的散播、減少感染ALV感染的亞臨床癥狀,減少該病帶來的危害。目前,多種植物多糖作為新型的添加劑被證明有抗病毒、抗腫瘤、抗氧化、抗輻射等多種生物學作用。本實驗室之前的研究發(fā)現(xiàn)泰山松花粉多糖(Taishan pine pollen polysaccharose,TPPPS)能顯著增強奇異變形桿菌和禽波氏桿菌亞單位疫苗的免疫效果,促使更強的免疫應答反應;提高獺兔的生產(chǎn)性能;對免疫抑制小鼠有免疫恢復作用。然而,前面所用TPPPS為水提醇沉法所提的總多糖,至今仍不清楚TPPPS中的有效活性成分及相應的生物學特性,因此限制了人們對TPPPS的認識及應用�；谝陨峡紤],本研究首先分析了TPPPS的成分和理化特性,并鑒定了每種成分的生物學活性;進一步對不同組份抑制B亞群禽白血病病毒(ALV-B)增殖的特性進行了分析;最終,通過建立感染感染ALV-B的雞免疫抑制模型,探究了TPPPS對ALV-B誘導的免疫抑制的免疫調(diào)理和恢復作用,為探索研發(fā)防制禽白血病的新途徑奠定基礎(chǔ)。本研究分以下三部分內(nèi)容:1.TPPPS部分理化特性和生物學活性的分析1.1 TPPPS的柱層析純化及各組分分子量和單糖組成的檢測為進一步研究TPPPS的功能和發(fā)揮作用的機理,本研究將粗提的多糖用DEAE纖維素柱和Sephadex G-200凝膠柱進一步的純化。結(jié)果顯示,松花粉多糖主要包括三種組分(分別命名為TPPPS1、TPPPS2和TPPPS3),并且分離得到了這三種組分的均一多糖。利用高效凝膠滲透色譜法(HPGPC)和高效液相色譜(HPLC)檢測三種多糖組分的分子量和單糖組成。結(jié)果表明TPPPS1、TPPPS2和TPPPS3的分子量分別為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構(gòu)成,TPPPS1-3組分之間各單糖組成的種類和比例存在明顯不同。以上研究結(jié)果提示這三種組份可能具有不同的生物學特性。1.2 TPPPS體外抗氧化性、免疫增強活性、抗病毒活性的檢測TPPPS各組分的分子量和單糖組成差異顯著,表明TPPPS1-3的生物學活性可能存在明顯的差異。為了進一步了解TPPPS的生物活性,本研究對TPPPS、TPPPS1、TPPPS2和TPPPS3的三種體外生物活性進行了檢測。結(jié)果表明,TPPPS2和TPPPS3顯著促進脾淋巴細胞的增殖,并且TPPPS3的效果強于TPPPS2;TPPPS3增強細胞因子白介素2(IL-2)和腫瘤壞死因子(TNF)的分泌,而TPPPS2僅可以顯著提高IL-2的分泌;值得注意的是,TPPPS1沒有以上作用,但誘導NO產(chǎn)生的效果最為顯著,表明TPPPS1具有最顯著的抗氧化活性;50μg/m L TPPPS3對ALV-B增殖作用最為顯著,并且作用的主要時期是在病毒的吸附期。本研究為使用TPPPS1-3合理比例的進一步研究可促進有效的免疫調(diào)節(jié)劑的開發(fā)和分析TPPPS的體外的抗病毒機制奠定了基礎(chǔ)。2.TPPPS體外抗病毒活性機制的分析2.1阻斷ELISA方法的建立為研究TPPPS對ALV-B增殖的顯著抑制主要發(fā)生在病毒吸附宿主細胞的過程中的機制,本研究根據(jù)ALV-B的吸附過程中起重要作用的兩個蛋白,病毒的囊膜糖蛋白gp85和宿主的受體蛋白tv-b,建立兩個ELISA方法。用原核表達的方法表達出gp85和tv-b蛋白,并制備這兩個蛋白的多克隆抗體,用這兩個蛋白和抗體建立兩個受體-病毒ELISA方法(方法I:gp85—tv-b—tv-b抗體—酶標二抗;方法II:tv-b—gp85—gp85抗體—酶標二抗)。用方陣試驗確定ELISA方法的各個條件,結(jié)果顯示方法I中g(shù)p85,tv-b,tv-b抗體的濃度,陰陽性臨界值分別是16μg/m L,32μg/m L,1:80,0.178,方法II中tv-b,gp85,gp85抗體的濃度分別是32μg/m L,32μg/m L,1:80,0.194,封閉液和TMB顯色時間都是5%脫脂奶粉和15 min。本試驗成功建立兩個ELISA方法用于分析TPPPS體外抗病毒活性機制。2.2 TPPPS體外抗病毒活性機制的分析利用建立的兩個受體-病毒ELISA方法,在gp85蛋白(或tv-b蛋白)包被ELISA板之后加入200μL不同濃度(100,101,102,103,104,105μg/m L)的TPPPS3溶液孵育,進行后續(xù)試驗觀察結(jié)果的變化。結(jié)果顯示,TPPPS3可以顯著降低ELISA方法I中的P/N值,對方法II的影響不顯著,這表明多糖能與病毒的gp85蛋白的結(jié)合,并且不能與tv-b受體蛋白結(jié)合,這種結(jié)合干擾了病毒與tv-b受體蛋白的結(jié)合,這是TPPPS抑制了病毒擴增的機制之一。本研究為進一步認識TPPPS在ALV-B吸附宿主細胞過程中的作用,為研究TPPPS抑制ALV-B在雛雞體內(nèi)增殖的機制奠定基礎(chǔ),為TPPPS在禽白血病防制方面的應用提供了依據(jù)。3.TPPPS對感染ALV-B雛雞免疫抑制模型的調(diào)理作用3.1感染ALV-B雛雞免疫抑制模型的建立雞群感染ALV后可造成1-2%的死亡率(偶爾可達20%或更高),而大部分雞只有亞臨床感染,主要是表現(xiàn)是生產(chǎn)性能下降和免疫抑制。本研究選用50只1日齡雛雞感染ALV-B,分別在感染后1-9周監(jiān)測感染雞的各項生理指標的變化,建立免疫抑制模型,檢測指標包括:發(fā)病率與死亡率,體重與免疫器官指數(shù),抗ALV-B特異性抗體水平,γ干擾素(IFN-γ)與白介素2(IL-2)水平,T淋巴細胞增殖率,CD4+和CD8+淋巴細胞亞群比例等。結(jié)果顯示通過對以上指標的檢測未發(fā)現(xiàn)感染雞死亡,但其生長狀況顯著差于正常雞,并且免疫器官發(fā)育遲緩,免疫反應不明顯,淋巴細胞活性降低,CD4+和CD4+/CD8+顯著下降等。結(jié)果表明,感染雞的免疫系統(tǒng)受到顯著地免疫抑制,免疫抑制模型建立成功,為分析感染ALV-B雛雞的免疫反應變化提供了依據(jù)。3.2 TPPPS對免疫抑制模型雛雞的調(diào)理作用據(jù)報道TPPPS對環(huán)磷酰胺導致的免疫抑制小鼠有免疫恢復作用,本研究通過給感染ALV-B的免疫抑制模型雛雞頸部皮下注射TPPPS,分析多糖對免疫抑制雞的免疫調(diào)理作用,同時在雛雞一周齡后免疫新城疫病毒(NDV)疫苗,之后檢測各項免疫指標,用以分析TPPPS對感染ALV-B雛雞免疫抑制模型的調(diào)理恢復作用。研究結(jié)果顯示,TPPPS組雛雞的體重和免疫器官指數(shù),外周血淋巴細胞增殖率,CD4+淋巴細胞亞群和CD4+/CD8+比值,IL-2和IFN-γ的分泌,和ALV-B的抗體陽性率都顯著增加,并且劑量為400 mg/kg(每千克雞)的TPPPS的效果是最顯著的。此外,在400 mg/kg TPPPS組抗新城疫病毒的抗體滴度也顯著高于其它試驗組。這些結(jié)果表明,TPPPS可以用作一種免疫增強劑,以減輕ALV-B造成的免疫抑制現(xiàn)象,為防制禽白血病提供新的思路和途徑。
[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.

【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S853.7

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