Characterization of the self-assembly of New Jersey polyomavirus VP1 into virus-like particles and the virus seroprevalence in Japan

浜松医科大学博士（医学）doctoral医学系研究科New Jersey polyomavirus (NJPyV) was discovered in 2014 in a pancreatic transplant recipient’s vascular endothelial cells. Here, in the recombinant baculovirus system, VP1 protein of NJPyV expressed in insect cells was processed. The protein self-assembled into virus-like particles (NJPyV-LPs) in a cell-type-dependent manner, and the particles were then released into the culture media. Spherical ~50-nm-dia. NJPyV-LPs of uniform size with morphology resembling that of the native particles of polyomaviruses were purified from the fraction at 1.33 g/cm3 in supernatants of VP1-expressing Sf9 cells. We investigated the antigenic properties of purified NJPyV-LPs and performed a VLP-based enzyme immunoassay to determine the age-specific prevalence of NJPyV infection in a general Japanese population aged 1–70 years. The overall seropositivity rate of anti-NJPyV antibodies was only 1.8%. This might be explained by the low circulation of NJPyV in Japan. This is the first report of a large-scale serological survey of NJPyV in Asia (n=1,050).doctoral thesi

Study on the Liquid Fermentation and Preparation of Anti-tumor Drug candidate Deacetylmycoepoxydiene

近年来，随着药物研究热点的转移，新药的开发已经拓展到了生态和物种较陆地生境更为复杂多样的海洋。海洋微生物特殊的生境，决定了其具有独特的代谢途径和防御机制。红树植物生长于海岸潮间带，其独特的生长环境赋予它丰富的微生物资源，已经成为近些年来备受瞩目的药用新资源。 本论文研究了海洋抗肿瘤候选新药去乙酰真菌环氧乙酯(Deacetylmycoepoxydiene，DAM)的液体发酵及提取制备工艺。DAM是红树植物内生真菌A-1-2-3(Phomopsissp.A-1-2-3)产生的一种骨架新颖的环氧二烯类化合物，具有较好的细胞毒作用。经动物试验抗肿瘤活性测定，该化合物对乳腺癌、胃癌等实体瘤具有显著的抑...The hot spot for searching novel drugs has been focused on the much more complex marine resources in recent years. Because of their special living conditions, marine microorganisms often produce various bioactive substances with novel functions and structures. Mangrove plants, as important new resources for potential pharmaceutical because of their ecosystems that straddled the land and the sea,...学位：理学硕士院系专业：生命科学学院生物学系_微生物学学号：2162007115205

Characterization of the self-assembly of New Jersey polyomavirus VP1 into virus-like particles and the virus seroprevalence in Japan

浜松医科大学博士（医学）doctoral医学系研究科thesi

Purify Deacetylmycoepoxydiene with Solventing-out Crystallization

为考察溶析结晶纯化去乙酰真菌环氧乙酯的效果,研究了不同的溶剂、反溶剂含量和导入方式对去乙酰真菌环氧乙酯结晶的影响.在静态结晶实验的基础上,通过设计正交实验l9(34)研究动态结晶过程中各因素对去乙酰真菌环氧乙酯收率和纯度的影响.结果表明:有效地控制反溶剂含量及其导入方式可以提高晶体的纯度和收率并影响晶体的形态.去乙酰真菌环氧乙酯动态结晶最佳条件:结晶温度为15℃,溶剂-反溶剂体积比(丙酮-水)为2∶3,反溶剂流加时间为3 H.In order to purify deacetylmycoepoxydiene(DAM)using solventing-out crystallization technique,the optimal conditions of crystallization were investigated by orthogonal design,and the recovery and purity of DAM were used as major evaluating contents.The results showed that the shapes of DAM crystal were influenced by different solvents,the recovery and purity of DAM were also influenced by the volume of anti-solvent and the way it was conducted.The optimal parameters are described as follows:crystallization temperature is 15 ℃,V(acetone)∶V(water)=2∶3,the flowing time of anti-solvent is 3 hours.国家海洋863项目(2007AA091503

Preparation of Deacetylmycoepoxydiene with Reversed Phase Chromatography

研究不同条件下反相柱层析对去乙酰真菌环氧乙酯分离纯化的影响.通过rP-18中压柱层析对从红树林内生真菌拟茎点霉(PHOMOPSISSP.)A-1-2-3诱变后突变株g1-1固体发酵提取物中的去乙酰真菌环氧乙酯进行了纯化精制.对rP-18柱层析纯化去乙酰真菌环氧乙酯操作条件进行了考察,使去乙酰真菌环氧乙酯的含量从28%左右提高到81.5%左右,回收率大于90%.采用rP-18直接洗脱,溶析结晶,重结晶,结晶残液rP-18分步洗脱的总工艺路线,去乙酰真菌环氧乙酯的总回收率为87.7%,产品最终纯度达到99%以上.The effects on purification of deacetylmycoepoxydiene(DAM) in different conditions were studied.Preparation of DAM from the crude extract of fermentation of strain G1-1,a mutant strain of mangrove endophytic phomopsis sp.A-1-2-3,was conducted using C18-silica gel reversed phase chromatography at moderate pressure.In the process,the operating conditions of C18-silica gel chromatography were studied experimentally,then the purity of DAM reached about 81.5% from about 28% in the crude extract,and the recovery of DAM was over 90%.The whole process of preparation was as follow: direct elution with RP-18,solvent-out crystallization,recrystallization and segment elution of residue of crystallization with RP-18.Through the whole process flow,the general recovery of DAM was 87.7%,and the purity of DAM reached 99% or higher.国家海洋863项目(2007AA091503)资

Technology of Pretreating Fermentation Broth of Deacetylmycoepoxydiene

为了降低去乙酰真菌环氧乙酯发酵液粘度,改进过滤效果,考察了β-葡聚糖酶对去乙酰真菌环氧乙酯发酵液粘度的影响,并进一步研究了不同类型的絮凝剂对发酵液过滤速度、沉降率、有效过滤体积和去乙酰真菌环氧乙酯含量的影响.通过正交试验,确定了非离子型聚丙烯酰胺的最佳絮凝工艺条件,应用统计学手段获得了最佳酶促反应时间和酶用量.结果表明,β-葡聚糖酶和非离子型聚丙烯酰胺的联合使用,可使滤速提高80%,有效过滤体积增加50%,保证了去乙酰真菌环氧乙酯的发酵量.In order to lower the viscosity of deacetylmycoepoxydiene fermentation broth and improve the effect of filtering,the impact of β-glucanase on the viscosity of DAM fermentation broth was studied.The effects of different types of flocculants on filtration rate of fermentation-broth,sedimentation rate,the effective filter volume and concentration of DAM were further studied.Through orthogonal test,the optimal flocculation conditions of the non-ionic flocculants were obtained,and enzymatic reaction time and enzyme dosage of β-glucanase were determined by statistical methods.The results show that the filtration rate is increased by 80% and effective filter volume is increased by 50% through the joint use of the β-glucanase and non-ionic flocculants.It ensures the fermentation volume of DAM.国家海洋863项目(2007AA091503)资

Scale-up of Deacetylmycoepoxydience Fermentation and the Controls of the Dissolved Oxygen Parameters for the Fermentation

报道了拟茎点霉P2-139(PHOMOPSIS SP.P2-139)在前期培养基质优化的基础上,进行的10,50和200 l发酵罐中培养条件和中试放大的研究结果.在10 l发酵罐中,P2-139菌株的最佳发酵培养基为:马铃薯240 g/l,葡萄糖75 g/l,陈海水20%(体积分数),PH自然.机械剪切力对去乙酰真菌环氧乙酯的产生和产量影响不大.临界氧浓度和klA测定结果表明,P2-139菌株生长的溶氧浓度(dO)值应维持在20%--30%以上才能保证菌株的正常生长和代谢;200 l罐中试放大dO值的控制为:发酵初期(0--96 H)20%以上,发酵中期(96--216 H)28%以上,发酵后期30%--60%;发酵基质为:马铃薯180 g/l,葡萄糖60 g/l,陈海水20%(体积分数),PH自然;发酵后期流加葡萄糖使残糖质量浓度控制在10 g/l左右.在此条件下,去乙酰真菌环氧乙酯的产量可达120 Mg/l以上.Phomopsis sp.P2-139 is a high-yield strain which produces deacetylmycoepoxydience,an anti-tumor compounds,commonly known as the Nanqiangjunsu.The paper reported the fermentation of the compound in 10,50 and 200 L bioreactor,respectively.In 10 L bioreactor,the P2-139 strain′s optimum fermentation medium component as followed(grams per liter):potato,240 g/L,glucose,75 g/L,and aged seawater,20%(by volume),pH free.There was litter effect of mechanical shear on the generation and production of deacetylmycoepoxydience.Limited oxygen concentration(DO) and KLa measurements showed that,dissolved DO must maintain at 20% to 30% or more in order to ensure the normal growth and metabolism of the P2-139 strain.In 200 L scale-up fermentation,the DO should be controlled as followed:more than 20% in the initial(0 h to 96 h),more than 28% in the metaphase(96 h to 218 h) and 30% to 60% in the later stage when the strain was cultured in potato 180 g/L,glucose 60 g/L,aged seawater 20%(by volume) and pH free.In the later stage of fermentation,glucose concentration was controlled at 10 g/L by method of fed-batched.Under these conditions,the yield of deacetylmycoepoxydience would reach 120 mg/L or more.国家高技术研究发展计划(863计划)项目(2007AA091503