【作者】 叶茂青；

【导师】 邹国林；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 真菌Cordyceps sinensis中文名为冬虫夏草菌,分类学上属于麦角菌科（Clavicipitaceae）,虫草属（Cordyceps（Fr.）Link）。C.sinensis是一种生长在特殊的环境中,具有独特生活史的药用真菌。C.sinensis寄生在蝙蝠蛾科昆虫的幼虫体内与其它一些真菌形成的复合体为我国民间较常用的名贵中药,称为冬虫夏草,别名虫草、冬虫草。冬虫夏草对生长条件要求极严而其生长环境特殊,现在尚无法大规模人工培养,而天然的冬虫夏草目前远远不能满足医疗市场的需要。C.sinensis菌丝体的成分与天然冬虫夏草的组成成分有一定的相似,初步研究表明其具有增强免疫,抗肿瘤和抗菌的活性。因此人工液体培养的C.sinensis菌是一个公认的非常有前途的中药冬虫夏草的代用品。 采用正交实验法,选择使用大豆粉和玉米粉作为C.sinensis菌丝体液体培养的主要原料,研究了C.sinensis的大规模液体培养的条件,以期为对C.sinensis蛋白药理活性的研究和C.sinensis菌丝体的大规模生产打下基础。正交实验结果表明,在使用大豆粉和玉米粉为主要培养原料时,C.sinensis的最佳培养基主要成分的配比为:大豆粉2%,玉米粉4%,蔗糖4%,KH₂PO₄0.05%,维生素B₁3mg/L。在此最佳配比条件下,培养基的起始pH值应为6.0左右的弱酸性环境,在培养过程中应充分满足培养时菌丝体的耗氧量,培养菌丝体的时间在4-5d为宜。 使用生化技术从C.sinensis菌丝体中提取了C.sinensis粗蛋白（主要为可溶蛋白部分）,并将C.sinensis粗蛋白分离成了5个组分（CS1、CS2、CS3、CS4、CS5）。建立了对蛋白样品的数个药理活性的检测方法(凝集素活性、抗肿瘤活性、核糖更多还原

【Abstract】 Cordyceps sinensis （Berk.） Sacc. is a caterpillar fungus of genus Cordyceps （Clavicipitaceae; Hypocreales; Ascomycotina）. This species endophytically parasitizes on dead caterpillars of the moth hepilus spp. Spores of C. sinensis geminate inside the caterpillars, filling the caterpillars with hyphae, and produce a stalked fruiting body. For centuries, the fruiting bodies of C. sinensis have been used as both food and tonic herbal medicine, named Dong-Chong-Xia-Cao in mandarin Chinese, which has been used to treat a variety of diseases. Due to being not cultured artificially on a large scale, native Dong-Chong-Xia-Cao can not supply medical marketable requirement. There are some same components between C. sinensis and native Dong-Chong-Xia-Cao, and pilot study has showed that C. sinensis have the activities of enhance immunity, antitumour and antimicrobial. Now, artificially cultured C. sinensis hypha has been seen as one of the best promising substitutes for herbal medicine Dong-Chong-Xia-Cao.Using the orthogonal experimental design, we studied the condition of large-scale culture medium by using soybean flour and corn flour as the main materials. Results of orthogonal experimention indicate that the best compositive proportion of the culture medium was soybean flour 2%, corn flour 4%, sucrose 4%, KH₂PO₄ 0.05 %and vitamin B₁ 3mg/ml. In this culture medium condition, the best pHof the culture medium was 6.0. In the culture period, providing enough oxygen was necessary, and the fitting culture time was 4-5 day.We obtained the crude protein of C. sinensis by using biochemistry techniques, and divided the crude protein of C. sinensis into five fractions （CS1,CS2,CS3,CS4,CS5） by gel filtration chromatogram. We established several methods for detecting the biological functions of protein in our lab （lectin, antitumour, RNase, DNase, proteinase, proteinase inhibitor, antimicrobial）, and detected the biological functions of C. sinensis protein by these methods. Experimental results indicated that the C. sinensis protein had not lectin, RNase and proteinase inhibitor activity, but the C. sinensis protein had antitumour, DNase, proteinase and antimicrobial activity. More experiments elucidated that C. sinensis protein could inhibit the growth of human cervix cancer （Hela cell） and liver cancer cell, and induce Hela cell apoptosis; C. sinensis protein had antibacterial activity, but had not effect on fungus; C. sinensis could excrete a proteinase into culture medium which also had fibrinolytic activity; C. sinensis mycelium contained at least one kind of acid DNase.A new acid deoxyribonuclease （DNase） was purified from the cultured mycelia of Cordyceps sinensis, designated CSDNase. CSDNase was purified by (NH^SC^ precipitation, gel filtration of Sephacryl S-100 HR, weak anion-exchange HPLC, and gel filtration HPLC. The protein was single-chained, with an apparent molecular mass of approximately 34 kDa revealed by SDS-PAGE, and its isoelectric point of 7.05 was estimated by isoelectric focusing. CSDNase acted on both double-stranded （ds） and single- stranded （ss） DNA, but acted preferentially on dsDNA. The optimum pH of CSDNase was pH 5.5 and the optimum temperature was 55 "C. The activity of CSDNase was not dependent on divalent cation, but it’s enzyme activity could be inhibited by high ion concentration. CSDNase hydrolyzes DNA, generating a terminus with 3’-phosphate and 5’-OH. These results indicated that the nucleolytic properties of CSDNase were essentially the same as other acid DNases that have been well characterized, and CSDNase was in fact a member of the acid DNase family. To our knowledge, this is the first report of an acid DNase was found in fungus.A resonance mirror biosensor with a stirred cuvette had been used to monitor theinteraction of immobilized CSDNase, bovine pancreas DNase I（Bp-DNase I） and porcine spleen DNase II（Ps-DNase II） with different DNA substrates in different condition. The association and dissociation rate constant of the three DNase interact with DNA were investigated at different temperatures, and the kinetics data （K^, ^diss） and equilibrium data （Ka> Ko） were evaluated. The thermodynamics parameters, changes in enthalpy （AH） and entropy （TAS）, were also evaluated from equilibrium data. The results indicated that the process of all three DNase interact with DNA was an entropy driving process. It appears that hydrophobic and electrostatic forces may be both involved in the binding process. High ion intensity could reduce the interaction of CSDNase and DNA, but too low ion intensity could also reduce the interaction. We had studied Mg2+ influence on the interaction between Bp-DNase I and DNA. At the same condition, Mg2+ could remarkably enhance the capacity of Bp-DNase I associated with DNA.Acid DNase was found mainly consisted in lysosome, which can keep function in a broad pH condition. The change of pH could change the structure of acid DNase, and regulate the biological function of the enzyme. Pilot study also indicated that acid DNase would transmembrane several times before it matured, which may pass through several structural transform. So, it is very important to study the protein folding of acid DNase in different conditions. Bovine immunoglobulin G （blgG） is a protein which is easy to be obtained. We studied the thermal denaturation, isothermal chemical and thermal chemical denaturation of blgG by differential scanning calorimetry （DSC） and fluorescence spectroscopy. We have established a method to study the protein folding process in our lab, that make good base for the research of the protein folding of acid DNase in future.更多还原