金龟子绿僵菌中苦马豆素的提取及其对新城疫疫苗免疫效果的影响
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摘要
目的旨在改进从金龟子绿僵菌发酵液中提取分离苦马豆素的方法,以期为增加苦马豆素的来源、降低苦马豆素临床应用成本,实现苦马豆素的药用价值,为人类和动物疾病的研究和有效治疗提供依据。
方法①苦马豆素提取工艺研究。定期用马铃薯葡萄糖琼脂(PDA)培养基对金龟子绿僵菌进行保藏、传代,用改良的查氏(Czapek’s)培养基发酵培养,培养条件为,温度25℃～27℃、pH 6.5～6.9﹑湿度45 %～55 %﹑培养14～16 d,获得金龟子绿僵菌发酵液。对发酵液进行预处理,采用萃取、吸附柱色谱、减压升华等方法提取分离出苦马豆素粗品。采用薄层色谱、气相色谱等方法对提取物进行初步鉴定分析;②金龟子绿僵菌发酵液提取物的体外抑菌活性试验。采用琼脂扩散法和试管法,以大肠埃希菌(Escherichia coli),金黄色葡萄球菌(Staphylococcus aureus),无乳链球菌(Streptococcus agalactiae),李斯特菌(Listeria monocytogenes),蜡状芽孢杆菌(Bacillus cereus)作为待检菌种,检测不同提取物的抑菌活性,从定性和定量二个角度对金龟子绿僵菌提取物的抑菌活性进行初步研究;③将100只14日龄健康无免疫雏公鸡随机分为5组,第Ⅰ一Ⅲ组分别注射高、中、低(4、2、1 mg/kg)3种剂量的金龟子绿僵菌次级代谢产物;IV组和V组分别为无佐剂组对照组(No Adjuvant, NA)和空白对照组。第Ⅰ一IV组动物于14日龄以新城疫病毒(NDV) LaSota株弱毒活疫苗滴鼻进行第1次免疫,2周后加强免疫1次。分别于首免前和首免后连续6 W在各免疫组随机抽取10只鸡检测血清中ND-HI抗体效价;于每次免疫后24 h检测外周血中白细胞数;于免疫后42日龄检测免疫器官指数;用MTT法检测脾脏淋巴细胞的增殖活性。
结果①马铃薯葡萄糖琼脂(PDA)培养基适用于金龟子绿僵菌的保藏、传代、分离与纯化;金龟子绿僵菌在改良的查氏(Czapek’s)培养基发酵培养上生长状态良好;从金龟子绿僵菌发酵液提取分离到白色粉末,经薄层色谱、气相色谱鉴定分析初步确定为苦马豆素;②6种金龟子绿僵菌发酵液的提取物对实验菌种均有一定程度的抑菌活性;③金龟子绿僵菌次级代谢产物剂量为1-2mg/kg,试验组鸡的抗体效价与雏鸡脏器(胸腺,脾脏,法氏囊)指数及外周血中白细胞数都显著高于对照组(P<0.05)。金龟子次级代谢产物单独或其协同脂多糖(LPS),植物血凝素P(PHA-P)刺激时,鸡脾脏淋巴细胞的增殖活性增强。金龟子绿僵菌次级代谢产物剂量为4mg/kg时试验组鸡的抗体效价,免疫器官指数和外周血中的白细胞数均减少;鸡脾脏淋巴细胞的增殖活性降低。
Subject: The purpose is seeking a effective technique for the extraction and isolation swainsonine from culture solution of Metarhizium anisopliae,searching for a new source of swainsonine, reducing clinical application cost of swainsonine, and realize swainsonine officinal value. Offering new theory base to effective therapy and research for hunman and animal disease.
Methods:①Research on the technical of swainsonine extraction . Metarhizium anisopliae was preserved, passaged,isolated and purified on potato dextrose agar (PDA)medium in a stated time, Inoculating Metarhizium anisopliae on Czapek′s medium, the culture conditions is that: temperture is 25～27℃,pH is 6.5～6.9 ,comparative humidity is 45%～55%,culture time is 14～16 days.Then Metarhizium anisopliae culture solution was obtained and pretreated. Swainsonine was isolated from culture solution by using the method of extraction, column chromatography, decompression sublimation. Production was identified by TLC and GC method.②Experimental study on antimicrobial effects of extractives from Metarhizium anisopliae in vitro. In order to study the bacteriostatic activity of extractives from Metarhizium anisopliae, agar diffusion and tube dilution method were used to observe its bacteriostatic efficacy in vitro.Choosing the five common pathogenic bacterium to obtain the different kinds of extraction antibacterial effect in vitro.③100 14-day-old chickens were randomly assigned into equal five groups. the chickens in three experimental groups were intramuscularly injected respectively with Secondary metabolites of Metsrhizium anisopliae at high, mediumand, low dose and vaccinated with Newcastle disease vaccine. two control groups were administrated with no adjuvant(NA) and normal group. The 1 st to 4rd four groups were immunized with the LaSota live vaccine against Newcastle disease virus(NDV)for two times with 14 days interval. Before and on days continuous 6 weeks after vaccination, 10 chickens were sampled randomly from each group for the dynamic change of serum HI antibody were determined. cellular numbers of leucocyte in peripheral blood were detected, On days 42 after vaccination, 5 chickens per group were collected to the weights of whole body,spleen,bursa and thymus were weighted for calculation of immune organ index.Proliferative response of spleen lymphocyte was examined by MTT method.
Results:①Cultured transferring Metarhizium anisopliae on potato dextrose agar(PDA) medium is suitable; Metarhizium anisopliae grew well on modified Czapek′s medium; Using gas chromatography(GC) could detecte swainsonine that in white powder which was isolated from fermentatoin broth.;②The result indicate that the five different pathogenic bacteriums was suppressed by all the 6 kinds of extractives from Metarhizium anisopliae at some level;③The secondary metabolites of Metarhizium anisopliae at dosage of 1-2 mg/kg could increased antibody titer,organ index and cellular number in blood. The proliferative activity of spleen lymphocyte was promoted by secondary metabolites of Metsrhizium anisopliae alone or being combined with LPS and PHA-P.The cellular number in blood,organ index,antibody titer and the proliferative activity of lymphocyte were decreased at the dosage of 4mg/kg.
方法①苦马豆素提取工艺研究。定期用马铃薯葡萄糖琼脂(PDA)培养基对金龟子绿僵菌进行保藏、传代,用改良的查氏(Czapek’s)培养基发酵培养,培养条件为,温度25℃～27℃、pH 6.5～6.9﹑湿度45 %～55 %﹑培养14～16 d,获得金龟子绿僵菌发酵液。对发酵液进行预处理,采用萃取、吸附柱色谱、减压升华等方法提取分离出苦马豆素粗品。采用薄层色谱、气相色谱等方法对提取物进行初步鉴定分析;②金龟子绿僵菌发酵液提取物的体外抑菌活性试验。采用琼脂扩散法和试管法,以大肠埃希菌(Escherichia coli),金黄色葡萄球菌(Staphylococcus aureus),无乳链球菌(Streptococcus agalactiae),李斯特菌(Listeria monocytogenes),蜡状芽孢杆菌(Bacillus cereus)作为待检菌种,检测不同提取物的抑菌活性,从定性和定量二个角度对金龟子绿僵菌提取物的抑菌活性进行初步研究;③将100只14日龄健康无免疫雏公鸡随机分为5组,第Ⅰ一Ⅲ组分别注射高、中、低(4、2、1 mg/kg)3种剂量的金龟子绿僵菌次级代谢产物;IV组和V组分别为无佐剂组对照组(No Adjuvant, NA)和空白对照组。第Ⅰ一IV组动物于14日龄以新城疫病毒(NDV) LaSota株弱毒活疫苗滴鼻进行第1次免疫,2周后加强免疫1次。分别于首免前和首免后连续6 W在各免疫组随机抽取10只鸡检测血清中ND-HI抗体效价;于每次免疫后24 h检测外周血中白细胞数;于免疫后42日龄检测免疫器官指数;用MTT法检测脾脏淋巴细胞的增殖活性。
结果①马铃薯葡萄糖琼脂(PDA)培养基适用于金龟子绿僵菌的保藏、传代、分离与纯化;金龟子绿僵菌在改良的查氏(Czapek’s)培养基发酵培养上生长状态良好;从金龟子绿僵菌发酵液提取分离到白色粉末,经薄层色谱、气相色谱鉴定分析初步确定为苦马豆素;②6种金龟子绿僵菌发酵液的提取物对实验菌种均有一定程度的抑菌活性;③金龟子绿僵菌次级代谢产物剂量为1-2mg/kg,试验组鸡的抗体效价与雏鸡脏器(胸腺,脾脏,法氏囊)指数及外周血中白细胞数都显著高于对照组(P<0.05)。金龟子次级代谢产物单独或其协同脂多糖(LPS),植物血凝素P(PHA-P)刺激时,鸡脾脏淋巴细胞的增殖活性增强。金龟子绿僵菌次级代谢产物剂量为4mg/kg时试验组鸡的抗体效价,免疫器官指数和外周血中的白细胞数均减少;鸡脾脏淋巴细胞的增殖活性降低。
Subject: The purpose is seeking a effective technique for the extraction and isolation swainsonine from culture solution of Metarhizium anisopliae,searching for a new source of swainsonine, reducing clinical application cost of swainsonine, and realize swainsonine officinal value. Offering new theory base to effective therapy and research for hunman and animal disease.
Methods:①Research on the technical of swainsonine extraction . Metarhizium anisopliae was preserved, passaged,isolated and purified on potato dextrose agar (PDA)medium in a stated time, Inoculating Metarhizium anisopliae on Czapek′s medium, the culture conditions is that: temperture is 25～27℃,pH is 6.5～6.9 ,comparative humidity is 45%～55%,culture time is 14～16 days.Then Metarhizium anisopliae culture solution was obtained and pretreated. Swainsonine was isolated from culture solution by using the method of extraction, column chromatography, decompression sublimation. Production was identified by TLC and GC method.②Experimental study on antimicrobial effects of extractives from Metarhizium anisopliae in vitro. In order to study the bacteriostatic activity of extractives from Metarhizium anisopliae, agar diffusion and tube dilution method were used to observe its bacteriostatic efficacy in vitro.Choosing the five common pathogenic bacterium to obtain the different kinds of extraction antibacterial effect in vitro.③100 14-day-old chickens were randomly assigned into equal five groups. the chickens in three experimental groups were intramuscularly injected respectively with Secondary metabolites of Metsrhizium anisopliae at high, mediumand, low dose and vaccinated with Newcastle disease vaccine. two control groups were administrated with no adjuvant(NA) and normal group. The 1 st to 4rd four groups were immunized with the LaSota live vaccine against Newcastle disease virus(NDV)for two times with 14 days interval. Before and on days continuous 6 weeks after vaccination, 10 chickens were sampled randomly from each group for the dynamic change of serum HI antibody were determined. cellular numbers of leucocyte in peripheral blood were detected, On days 42 after vaccination, 5 chickens per group were collected to the weights of whole body,spleen,bursa and thymus were weighted for calculation of immune organ index.Proliferative response of spleen lymphocyte was examined by MTT method.
Results:①Cultured transferring Metarhizium anisopliae on potato dextrose agar(PDA) medium is suitable; Metarhizium anisopliae grew well on modified Czapek′s medium; Using gas chromatography(GC) could detecte swainsonine that in white powder which was isolated from fermentatoin broth.;②The result indicate that the five different pathogenic bacteriums was suppressed by all the 6 kinds of extractives from Metarhizium anisopliae at some level;③The secondary metabolites of Metarhizium anisopliae at dosage of 1-2 mg/kg could increased antibody titer,organ index and cellular number in blood. The proliferative activity of spleen lymphocyte was promoted by secondary metabolites of Metsrhizium anisopliae alone or being combined with LPS and PHA-P.The cellular number in blood,organ index,antibody titer and the proliferative activity of lymphocyte were decreased at the dosage of 4mg/kg.
引文
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