【作者】 王芙蓉；

【导师】 雷朝亮；

【作者基本信息】 华中农业大学 ， 动物学， 2008， 博士

【摘要】 家蝇是一种世界性分布的昆虫,繁殖系数高,生活周期短,养殖技术简单,被国际上列为新型蛋白源昆虫之首。家蝇从幼虫到成虫均生活在杂菌孳生的环境里,蛆体表面及体内含有大量的病菌,能在人类和动物间传播多种疾病,它们极强的抗病能力成为了科学家们研究的热点。本文以家蝇幼虫为研究对象,从家蝇幼虫体内提取了生物活性蛋白物质,并研究了其抗病毒、抗氧化和护肝作用,主要研究结果如下:1.家蝇幼虫组织匀浆液的抗病毒作用在体外,采用鸡胚法和乙型肝炎病毒表面抗原诊断试剂盒检测了家蝇幼虫组织匀浆液的抗病毒活性。实验结果表明:不同浓度的家蝇幼虫组织匀浆液对流感病毒在鸡胚内的增殖都有显著的抑制作用。试剂盒的检测结果也说明:家蝇幼虫组织匀浆液在体外对乙肝病毒表面抗原有很好的破坏作用,其破坏率可达87.5%。2.家蝇幼虫活性蛋白的提取、分离纯化及活性研究以脱脂蝇蛆粉为材料,根据蛋白质溶解性的不同,从家蝇幼虫体内提取了5类蛋白质,并分别研究了其抗病毒和抗氧化的活性。对其中含量高并且抗氧化和抗病毒活性好的组分进行了进一步的分离,并且确定了最佳的制备工艺。采用阳离子交换色谱将蛋白质提取物进一步分离并检测了各组分的抗病毒活性。研究结果表明:脱脂蝇蛆粉中清蛋白的含量最高,并且抗氧化和抗病毒活性最好,以此为目的蛋白,进一步分离得到蛋白质提取物。蛋白质提取物的最佳提取条件为pH 7.0,盐浓度0.18mol/L。从蛋白质提取物中分离出4个蛋白质组分A1-4,抗病毒活性显示,这4个组分对流感病毒有一定的抑制作用,但活性与蛋白质提取物相比没有提高。3.蛋白质提取物抗新城疫病毒的活性采用鸡胚的方法研究了蛋白质提取物对新城疫病毒在鸡胚内增殖的抑制作用。毒性实验表明:给鸡胚注射不同浓度的蛋白质提取物后,鸡胚存活率均为100%。说明蛋白质提取物对鸡胚的生长发育没有影响,基本上不产生毒性作用。抗病毒实验结果表明:蛋白质提取物的各个剂量组均能不同程度的抑制新城疫病毒在鸡胚内的增殖。4.蛋白质提取物抗脊髓灰质炎病毒的活性采用细胞培养的方法,体外检测了蛋白质提取物对病毒感染细胞的抑制作用。通过终点稀释法研究了蛋白质提取物与病毒在体外中和后,对病毒的抑制作用;检测了蛋白质提取物对100个TCID₅₀病毒的最低抑制浓度,并计算治疗指数。结果表明:蛋白复合物对Hela细胞的半数有毒浓度CC₅₀为331.13μg/mL。蛋白质提取物与病毒直接作用后可使病毒的感染滴度下降两个数量级。对100个TCID₅₀脊灰病毒的治疗指数大于33,说明蛋白质提取物具有很好的抗脊灰病毒的作用。5.蛋白质提取物抗禽流感病毒的活性采用血凝的方法检测了蛋白质提取物在体外对禽流感H₉N₂病毒的杀灭作用。实验结果表明:蛋白质提取物对鸡血红细胞的毒性作用比较小,在5 mg/mL以下没有观察到毒性作用。蛋白质提取物与病毒混合一段时间之后进行血凝实验检测,病毒的血凝效价比病毒对照组显著降低。6.蛋白复合物抗疱疹病毒的活性采用细胞培养的方法,体外检测了蛋白质提取物在体外与病毒中和作用、病毒和蛋白质提取物同时加入以及先加蛋白质提取物后加病毒三种作用方式下蛋白复合物抗疱疹病毒的作用。结果表明:蛋白复合物对Vero细胞的半数中毒浓度CC₅₀为236.63μg/mL。在前两种作用方式下,蛋白复合物显示出很好的抗疱疹病毒的作用;而蛋白质提取物对细胞的保护作用检测结果发现,蛋白质提取物先与细胞作用一段时间后,对病毒的感染力基本上没有影响。该结果说明蛋白质提取物可能不是通过对细胞的保护作用起作用的。7.蛋白复合物抗人流感病毒的活性采用细胞培养的方法,让病毒感染细胞,并采用血凝的方法检测了病毒感染细胞后培养的上清液中病毒的滴度。实验结果表明:蛋白质提取物对MDCK细胞的半数中毒浓度CC₅₀为284.45μg/mL。不同浓度的蛋白质提取物在体外与病毒中和后,能显著地降低病毒的感染力,而阳性药物病毒唑的作用却不太明显。当药物与病毒同时加入时,蛋白质提取物与病毒唑对病毒在细胞内的增殖都起到了很好的抑制作用。蛋白质提取物和病毒唑对细胞的保护作用都不强,均不能有效地抑制病毒增殖。病毒唑对病毒感染的治疗作用比较强,而蛋白质提取物对病毒直接杀灭的能力比较强。这预示蛋白质提取物可能具有与病毒唑不同的作用机理。8.家蝇幼虫蛋白质提取物的抗氧化作用分别采用脱氧核糖-铁体系法以及邻苯三酚自氧化法测定了蛋白质提取物的清除羟基自由基和超氧阴离子自由基的作用。抗氧化实验结果表明:蛋白质提取物具有很好的抗羟基自由基和清除超氧阴离子自由基的作用。蛋白质提取物清除羟基自由基的IC₅₀为1.23 mg/mL。当蛋白质提取物浓度为34.63μg/mL时则能清除50%的超氧阴离子自由基,当浓度达到1 mg/mL时则能完全抑制超氧阴离子自由基的产生。9.家蝇幼虫蛋白质提取物的护肝作用以四氯化碳所致肝损伤为模型,从生化指标和病理组织两个方面研究了蛋白质提取物对四氯化碳导致的小鼠肝损伤的保护作用。实验结果发现,四氯化碳造模很成功。灌胃蛋白质提取物以后能显著地降低由四氯化碳所导致的AST和ALT的升高,提高肝脏合成蛋白质的能力。肝组织病理切片显示:四氯化碳损伤组与正常对照组相比,肝脏均表现出典型的中毒性肝损伤病理改变;而蛋白质提取物的三个剂量组肝脏形态与结构基本完好,没有出现细胞坏死。从以上生化指标和病理组织两个方面的结果可以看出:蛋白质提取物对四氯化碳诱导的肝损伤具有很好的保护作用。本研究的结果表明:从家蝇幼虫体内提取的蛋白质提取物具有很好的抗病毒、抗氧化和护肝作用,对人体没有毒副作用,具有很好的临床应用价值。更多还原

【Abstract】 Housefly（Musca domestica vicina M.） distributed all over the world.The propagation coefficient of housefly is very high and their life history is short.The technology to breed is very simple and they are listed as the top new type resource insect enriched of protein.Housefly lives in the condition of pathogen propagating and completes its life history in decaying organic material.There is a large number of bacterium in the larvae of housefly both in vitro and in vivo.They can spread various diseases between humans and other animals,while they can thrive without disease.Their strong disease-resisting abilities become a new topic of general interest.In this study, bioactive proteins were extracted from the larvae of housefly and their chemical composition was analyzed.Moreovr,their antioxidant,hepatoprotective and antiviral activities were also detected.The major results were as following:1.Antiviral activity of tissue homogenate of the housefly larvaeThe method of chicken embryo and the diagnostic reagent kit for surface antigen of Hepatitis B virus（HBV） were adopted to detect the antiviral activity of tissue homogenate of the housefly larvae in vitro.The results showed that different concentrations of tissue homogenate had significant effects on inhibiting the proliferation of influenza virus in chicken embryo.The results of the diagnostic reagent kit also indicated that the tissue homogenate had remarkable destructive effect on HBV and the destructive efficiency reaches 87.5%.2.Isolation and characteristics of active protein from the larvae of houseflyFive sorts of proteins were isolated from the body of the housefly larvae according to their solubility based on the defatted powder and their antioxidant activities and antiviral activities were measured.Further isolation was processed on the component with high content and well activity of antiviral and antioxidant.An optimal technology for preparation was determined.Further isolation was processed on the protein extraction by cation exchange chromatography and the antiviral activity of each fraction was detected. The results showed that albumin was the main component of the housefly defatted powder and it had better antioxidant and antiviral activities than others.Therefore,we further isolated and obtained the protein extraction.The optimal extracted condition for NaC1 0.18 mol/1 and pH value 7.Four fractions（A1-4） were isolated from the protein extraction.The antiviral results showed that all of the fractions inhibited the proliferation of influenza virus in some degree,but the effects had not been improved compared with the protein extraction.3.Antiviral activity against Newcastle disease virus（NDV） The method of chicken embryo was adopted to detect the depressive effect of the protein extraction on the proliferation of Newcastle disease virus in chicken embryo.The result of toxicity test showed that the survival rates of all group were 100%after injection of protein extraction with different concentrations.This indicated that protein extraction had no effect on the growth and development of chicken embryo and it had no toxicity.The antiviral results showed that the protein extraction inhibitted the proliferation of Newcastle disease virus in chicken embryo at different concentrations.4.Antiviral activity against Poliomyelitis virus（PV）The method of cell culture was adopted to detect the antiviral activity of protein extraction in vitro.End-point dilution method was adopted to detect the effect of protein extraction on Poliomyelitis virus after neutralization of protein extraction and virus in vitro.We also detected the minimum depressive concentration of protein extraction on 100个TCID₅₀ virus and the selectivity index（SI） was calculated.The MTT method indicated that protein extraction had minimal cytotoxicity against Hela cells and the CC₅₀ was 331.13μg/mL.The preliminary studies on the inactivation of PV-1 indicated that the loss of infectious capacity was more than two log（10） units when the virus suspensions were pre-incubated for 2 h with protein extraction prior to the cell infection.Protein extraction strongly elicit its antiviral activity against PV-1 in Hela cells with an EC₅₀ of 10μg/mL and the SI was calculated to be more than 33.All of the results indicated that protein extraction had antiviral activity against PV-1 virus.5.Antiviral activity against avian influenza virus（AIV）The interaction between virus and cell made it possible to detect anti-influenza virus agents on the basis of the modification of the HA test.The method of HA test was adopted to detect the antiviral activity of protein extraction on avian influenza virus H₉N₂ in vitro.The results showed that protein extraction had little toxic effect on red blood cells and no cytopathic effects were observed below the concentration of 5 mg/mL. Different doses of protein extraction exhibited,to some degree,inhibition of viral adsorption of RBC and the end point of haemagglutinin dilution was greatly decreased compared with the control.6.Antiviral activity against herpes simplex virus（HSV）The method of cell culture was adopted to detect the antiviral activity of protein extraction in vitro.Three actions were detected as follows:neutralization of protein extraction and virus in vitro,simultancous effect of protein extraction and viruses and protective effects of protein extraction on Vero cells.Protein extraction exhibit remarkable antiviral activities against herpes simplex virus on the first two effect patterns. While the result of protective effects on cells showed that protein extraction had no effect on the infectivity of virus.This suggested that the effect of protein extraction could not due to the protective effects on cells.7.Antiviral activity against influenza A virusThe method of cell culture was adopted to detect the antiviral activity of protein extraction on influenza A virus in vitro.HA test was used to detect the virus titer in the supernatant of the cells culture infected by influenza A virus.The MTT method indicated that protein extraction had minimal cytotoxicity against Vero cells and the CC₅₀ was 284.45μg/mL.Protein extraction remarkably depressed the infectivity of virus after the neutralization action with virus,which was better than the positive drug Ribavirin.Both protein extraction and Ribavirin significantly inhibited the proliferation of influenza A virus in MDCK cells when drugs and viruses acted simultaneously.Both protein extraction and Ribavirin had no protective effect on the MDCK cells and could not depress the proliferation of virus effectively.Ribavirin had strong therapeutic effect on the virus infection,while protein extraction had virucidal activity on influenza A virus. This might be related to the mechanism of their action.8.Antioxidant effects of protein extractionThe Fe³⁺-ascorbate-EDTA-H₂O₂ system and the auto-oxidation system of pyrogallol were adopted to detect the antioxidant activity of protein extraction.The antioxidant results showed that the protein extraction had good scavenging activities on both hydroxyl radical and superoxide anion radical.The 50%hydroxyl radical scavenging concentration of the protein extraction was 1.23 mg/mL.The 50%superoxide anion radical scavenging concentration of the protein extraction was 34.63μg/mL calculated from the dose-activity curves.At the concentration of 1 mg/mL,superoxide anion radicals were completely scavenged.9.Hepatoprotective effect of protein extractionThe protective effects of protein extraction on CCl₄-induced hepatotoxicity were studied from both biochemical measurements and morphological observations.The results showed that the model of hepatic injury was successful.The AST and ALT activities of serum were significantly decreased and the ability of hepatocytes to synthesis protein was greatly improved after treated with the protein extraction.Photomicrographs of haematoxylin-eosin stained liver tissue showed that the single administration of CCl₄ caused serious liver damage in all animals of this group.The hepatocytes around the central vein were complete necrosis and the cellular boundary was lost.No histological abnormalities were observed in rats of the control group and also the groups of administration of various doses of protein extraction pretreated with CCl₄.The histological changes associated with the hepatoprotective activity in three prescriptions of protein extraction supported the estimation of the biochemical results.In conclusion,the hepatoprotective effects of protein extraction from the larvae of M.domestica were demonstrated by the biochemical measurements and the morphological observations using the acute hepatic damage model.In conclusion,protein extraction isolated from the larva of housefly,had remarkable antioxidant,hepatoprotective and antiviral activity in vitro.It had no adverse reaction on our body and it had preliminary clinical and applicable value in medicine.更多还原