实时定量PCR构建法夫酵母内参基因β-actin、 gpd、18S rRNA标准品质粒和标准曲线

为建立检测法夫酵母JMU-MVP14中虾青素合成相关基因在不同生长时期表达水平的实时定量PCR方法,构建法夫酵母JMU-MVP14的管家基因β-actin、gpd、18S rRNA的标准质粒,进行实时定量PCR,制作标准曲线及回归方程.β-actin基因标准曲线相关系数（R2）=0.9956,扩增效率（E） =96.93％;gpd基因标准曲线相关系数（R2） =0.9901,扩增效率（E） =93.78％;18S rRNA基因标准曲线相关系数（R2） =0.9981,扩增效率（E）=98.76％.3个基因片段的熔解曲线均呈单峰;扩增曲线呈典型的S型动力学曲线,指数期和平台期明显,为理想的熔解曲线和扩增曲线.用geNorm软件对三个管家基因的稳定性进行分析,三个基因的稳定性排序为β-actin＞ 18S rRNA＞ gpd,故β-actin和18S rRNA较适合作为研究法夫酵母JMU-MVP14定量实验的内参基因.     

伞形科两种植物幼苗生长对光照强度的可塑性响应

将明党参（Changium smyrnioides Wolff）和峨参（Anthriscus sylvestris Hoffm．）幼苗置于按全光照百分率为100％（S100）、65％（S65）和25％（S25）的人工控制光环境下处理。结果表明,光照强度显著影响明党参和峨参幼苗的生长：明党参在S6,生长最好,株高、冠幅、叶长、叶宽、地上地下生物量等均达到最大值,且S65的明党参生物量是S100的2倍、S25的3倍左右;而峨参地上部生长随着光照强度的减弱而增强,S25生长最好,但地下部生长则在S65生长最好,表现出与明党参同样的响应规律。明党参在不同光照强度下生长速率差异显著,S65显著高于其余两个处理;峨参虽然也在S65生长速率最大,但在不同光照强度间无差异。明党参生长速率明显小于峨参,总生物量约为峨参的1／3—1／8,相对于峨参而言明党参是一个在幼苗阶段生长缓慢的物种。     

温度对双低两用核不育水稻96-5-2S与培矮64S育性的影响

在自然变温、人工控温及冷水灌溉条件下,比较研究了温度对双低两用核不育水稻96－5－2S与两用核不育水稻培矮64S育性影响的差异。结果表明：（1）当它们在雄性育性转换温敏感期1－12d平均自然日均温23.0-23.8℃的低温时,96－5－2S表现不良,套袋自交结实率为0,而培矮46S可育,套袋自交结实率为0．1％－4．5％;（2）在它们雄性育性转换温敏感期用22℃恒温处理5d,96－5－2S败育彻底,套袋自交结实率为0,而培矮64S可育,套袋自交结实率为10．7％;用17℃恒温处理6d,96－5－2S与培矮64S均可育,但96－5－2S套袋自交结实率（6．8％）显著高于培矮64S（2．5％）;（3）在它们雄性育性转换温和不同温度的冷水串灌15d,水深维持在20cm左右,当水温为22－22．5℃时,96－5－2S不育,结实率为0,而培矮64S可育,结实率为18．5％;当水温为19．5－21．5℃时,96－5－2S与培矮64S均可育,但96－5－2S结实率（2．5％－45．1％）显著或极显著低于培矮64S（50．4％－56．9％）以上结果说明：导致双低两用核不育水稻96－5－2S雄性不育的起点温度与导致其生理不育的下限温度均低,其不育性比培矮64S更稳定,耐寒性比培矮64S更强,即可确保制种安全,又可确保自身繁殖,对加快两系法杂交水稻的发展步伐将起到重要的促进作用。     

NisinZ的定点突变及突变体性质的研究

以本实验室构建的含nisZ基因的质粒pHJ201为模板,采用定点突变技术将乳链菌肽Z分子中B环第8位Thr突变为Ser（T8S）、将第2位Dhb突变为Dha和第31位His突变为Lys（T2S/H31K）以及将第27位Asn突变为Lys和第31位His突变为Lys（N27K/H31K）,以pMG36e为载体,电击转化乳酸乳球菌（L.lactis）NZ9800进行表达。对表达产物性质的研究结果表明,3个突变体的抑菌谱和溶解度未发生变化,其抑菌活性略有下降,但它们的稳定性表现各不相同：N27K/H31K的稳定性与NisinZ几乎一致,而T8S和T2S/H31K的稳定性有明显提高,在pH9条件下100℃加热5min仍不丧失抑菌活性。     

实时定量PCR评估金针菇的内参基因稳定性

金针菇在中国和日本是最受人喜爱的食用菌之一,在重要栽培食用菌中产销量排名第四。近年来,已在活性物质、分子标记及基因鉴定方面开展了大量工作,但未见有金针菇内参基因稳定性研究的报道,导致金针菇基因表达研究无内源参考基因稳定性数据作为参考。本研究采用geNorm、NormFinder、BestKeeper和RefFinder 4种软件评估18S核糖体RNA（18S）、28S核糖体RNA（28S）、60S核糖体蛋白L18（Rpl18）、肌动蛋白1（Act1）、3-磷酸甘油醛脱氢酶（Gapdh）、翻译延伸因子EF1-alpha（Ef1A）、DNA指导的RNA聚合酶亚基2（Rpb2）、细胞色素C氧化酶亚基1（Cox1）及细胞色素b（CytB）等9个内参基因的稳定性。综合分析结果显示,单个基因中Act1基因最稳定,CytB表达稳定性最差,但单个候选内参基因的稳定性不能够满足定量PCR实验要求;根据geNorm软件分析结果,CytB和Rpb2是金针菇定量PCR的稳定内参基因组合。本研究首次评估了金针菇的内参基因稳定性,将为后续的基因表达研究提供参考。     

马齿苋多糖对S180荷瘤小鼠免疫功能的影响

本文探讨马齿苋多糖对S180荷瘤小鼠免疫功能的影响。马齿苋采用水提醇沉法得到马齿苋多糖,分别以50、100、200mg／kg通过腹腔给药10d,观察马齿苋多糖对S180荷瘤小鼠的抑瘤作用及对小鼠淋巴细胞转化功能、腹腔巨噬细胞的吞噬能力、白介素-l（IL-1）和白介素-2（IL-2）生成量的影响。结果显示,马齿苋多糖对S180荷瘤小鼠有明显的抑瘤作用,抑瘤率分别为16．92％、51．45％和64．96％。不同剂量马齿苋多糖与对照组相比可明显促进淋巴细胞的转化、小鼠腹腔巨噬细胞吞噬能力,可有效的增加荷瘤小鼠脾淋巴细胞的转化和腹腔巨噬细胞的吞噬能力以及白介素-1（IL-1）和白介素-2（IL-2）的分泌。说明马齿苋多糖对S180荷瘤小鼠具有显著的抗肿瘤作用,其作用机制与增强小鼠免疫作用有关。     

基于新型冠状病毒S蛋白结构及入胞机制的抗体与药物研发

新型冠状病毒肺炎,世界卫生组织命名为“2019冠状病毒病”（corona virus disease 2019, COVID-19）,是一种由2019新型冠状病毒（2019 nCov）感染导致的肺炎。目前新冠肺炎在全球广泛流行,且疫情尚未得到全部控制。由于新型冠状病毒表面的刺突蛋白（spike protein,S）介导病毒与细胞膜受体结合并参与入胞过程,S蛋白在病毒的传播过程中发挥着重要作用。针对S蛋白的研究不仅可以解析病毒相关蛋白质结构与功能,阐释其入胞机制,同时也为新冠肺炎的预防、诊断与治疗提供相关信息,有着重要的应用价值。S蛋白与特异性受体--血管紧张素酶II（angiotensin converting enzyme II, ACE2）结合,相较于SARS病毒,新型冠状病毒S蛋白的RBD区域（receptor binding domain）与ACE2亲和力更高,但其S蛋白与ACE2结合能力整体上弱于SARS病毒。S蛋白结合ACE2受体 介导的新型冠状病毒入胞机制包括胞吞和非胞吞途径。丝氨酸蛋白酶2（transmembrane protease serine 2, TMPRSS2）、溶酶体组织蛋白酶（lysosomal cathepsin）和Furin蛋白酶可切割S蛋白S1和S2亚基间的酶切位点,促进病毒和靶膜的融合。基于S蛋白的结构,本文从抗体的结合位点、来源与类型等方面对靶向新型冠状病毒S蛋白的抗体进行了比较分析,对相关药物作用机制与进展进行了综述。虽然靶向冠状病毒S蛋白的抗体和药物特异性高,治疗效果较好,但部分试剂的作用机制、安全性、适用性和稳定性等性质仍未研究透彻,需要严格评估,因此其研发与应用也存在着一定挑战。     

间作密植和单行茶园节肢动物群落组成差异

1998年1－12月每月下旬,对皖南敬亭山茶场栗－茶间作,梨－茶间作,3行密植和单行条植茶园节肢动物群落的调查表明：植食性昆虫种数分别占各类型茶园总物种数51．9％,53．6％,54．4％和56．6％,个体数依次占各类茶园总个体数92．0％,93．5％,93．6％和95．0％,捕食性昆虫和捕食螨种数则分别占11．3％,10．0％,9．8％,10．9％,个体数占2．0％,1．1％,2．0％和1．5％,寄生性昆虫种数占9．2％,9．1％,9．3％,和9．3％,个体数占1．8％,1．1％,1．8％和1．3％。蜘蛛种数占24．7％,20．1％,22．4％和19．4％,个体数占3．5％,3．4％,2．1％和1．8％,4类茶园中的优势类群都是鳞翅目,同翅目,翅目,双翅目和蜘蛛目,月平均丰富度（S）和多样性指数H大小,栗－茶间作（S＝74,H＝1．33）,梨－茶间作（S＝49,H’＝1．24）,3行密植（S＝31,H’＝1．03）和单行条植茶园（S＝23,H’＝0．89）,主成分分析揭示了群落稳定性的大小：栗－茶间作＞梨－茶间作＞3行密植＞单行条植茶园。     

（S）-（-）-β-羟基苯丙酸乙酯的微生物法合成

采用酿酒酵母CGMCC No．2266菌体,不对称还原β-羰基苯丙酸乙酯制备光学纯（S）-（-）-β-羟基苯丙酸乙酯。结果表明：采用初始pH为8．0的液体发酵培养基培养的CGMCCNo．2266菌体经过50℃预热处理30min后用于生物转化获得的（S）-（-）-G-羟基苯丙酸乙酯对映体过剩值可以达到100％ee。确定了合成（S）-（-）-β-羟基苯丙酸乙酯的较佳转化条件为pH7．0,温度30℃,转化时间24h,底物浓度为3．63mmol／L,菌体用量为86g/L（干重／反应体积）。以10％葡萄糖为辅助底物,产率比不加辅助底物时提高了75．4％。在最佳转化条件下反应转化率及（S）-（-）-β-羟基苯丙酸乙酯对映体过剩值可分别达到98．4％和100％ee。     

重组青霉素G酰化酶拆分制备（S）-邻氯苯甘氨酸

对产青霉素G酰化酶的重组枯草芽胞杆菌发酵产酶条件进行优化,确定优化后的发酵条件：可溶性淀粉10g/L、蛋白胨12g/L、酵母粉3g/L、NaCl10g,／L;pH7．5、培养温度37℃、装液量80mL（500mL三角瓶）、培养28h,青霉素G酰化酶的表达水平由最初的7．34U／mL提高至18．23U／mL。以表达青霉素G酰化酶的枯草芽胞杆菌发酵液为酶源,在水相中对映选择性催化N-苯乙酰-（R,S）-邻氯苯甘氨酸制备（S）-邻氯苯甘氨酸,当底物浓度为100mol／L时转化4h,转化率达44．2％。对底物浓度为80mmoL／L反应液中的（S）-邻氯苯甘氨酸进行分离,达到理论收率的94．29％（以N-苯乙酰-（R,S）-邻氯苯甘氨酸的0．5倍摩尔量为理论产率）,e．e．值大于99．9％。170℃条件下,N-苯乙酰-（R）-邻氯苯甘氨酸与苯乙酸共熔消旋为N-苯乙酰-（R,S）-邻氯苯甘氨酸可用于循环拆分。     

治疗用A型肉毒毒素的制备及其质量控制

我国治疗用Ａ型肉毒毒素采用酸等电点沉淀,磷酸盐提取,核糖核酸酶处理,ＤＥＡＥ Ａ５０离子交换层析,硫酸铵浓缩及自然结晶等程序从Ａ型肉毒梭菌培养物中提取,并经稀释、冻干而成。它毒力强、纯度高、性能稳定,宜于长期保存。经生化、免疫学测定,毒素系神经毒素和血凝素的复合体,纯度为２５～３．０×１０７ＬＤ５０（小鼠,下同）／ｍｇｐｒ,ＯＤ２６０／ＯＤ２８０≤０．５５不仅达到了美国ＦＤＡ对注射用Ａ型肉毒毒素的质量要求,而且在冻干损失,稳定性方面还优于美国制品。     

Chemoenzymatic synthesis of both enantiomers of alpha-tocotrienol

The stereoselective acylation of the achiral chromanedimethanol derivative 1 by vinyl acetate in the presence of Candida antarctica lipase B gave the (S)-monoester 2 in high enantiomeric purity (ee > or = 98%). Enzymatic hydrolysis of diesters of compound 1 failed to give (R)-monoester 2 in good yield and high ee. Thus, both enantiomers of alpha-tocotrienol were synthesized from the (S)-monoester 2.     

基因工程变链菌防龋疫苗的研制

本文采用我们自己构建的基因工程龋齿疫苗菌株,大罐发酵培养３批、菌体收量平均湿重为２．６７ｇ／Ｌ,表达率为４４．２７％。菌体经超声裂解,硫酸铵和链霉素粗提,再经吸附层检,凝胶过滤和离子交换柱层析等步骤纯化精提ＧＴＦ抗原３批。提纯的ＧＴＦ抗原,平均比活,提纯倍数和收率,分别为５８．３４ｕ／ｍｇ、６．０８倍和１９．２５％。此抗原经ＳＤＳ－ＰＡＧＥ电泳,分子量为５５ＫＤ,纯度达到９６．７％,并对湿度具有较好的稳定性能。且此抗原制备的疫苗免疫家兔具有较好的免疫原性     

抗生殖器疱疹病毒卵黄免疫球蛋白(IgY)的研究

检测了鸡卵黄中抗生殖器疱疹病毒(HSV-2)抗体的产量、纯度、来源及稳定性。采用生殖器疱疹病毒(HSV-2)作为抗原免疫广州黄村鸡。通过改良水稀释法提取卵黄中的IgY。双紫外光波长测定抗体含量,SDS-PAGE电泳检测抗体纯度。Western blot免疫印迹法测定该抗体来源。ELISA检测IgY对温度、酸碱度的稳定性。结果,蛋黄液中抗体质量浓度13.6g.L-1,抗体纯度达96.2%。免疫印迹证明IgY与鸡血清中的IgG具有相同的分子量和抗原性。IgY具有良好的热稳定性,对酸碱具有一定的耐受力。WD水稀释法能得到高产量、高纯度的特异性IgY,而且有良好的生物学活性。     

Development and validation of a method for the purity determination of (3beta,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol(FF-MAS) in pharmaceutical products containing recombinant human albumin

A simple and sensitive method has been developed and validated for purity determination of FF-MAS (also known as (3beta,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol an endogenous substance usually present in the pre-ovulatory follicular fluid) at very low concentrations (200 ng per unit) in pharmaceutical formulations containing RECOMBUMIN (recombinant human albumin) as the matrix. The paper focuses on development of the sample preparation for the product containing recombinant human albumin. After removal of recombinant human albumin by precipitation using a mixture of water and ethanol, the FF-MAS was concentrated by evaporation using a vacuum centrifuge and the prepared sample was analyzed. The purity method was based on a reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet absorption detection at 250 nm. The method was validated according to ICH guidelines. The method indicated a significant degree of specificity with good selectivity and no significant effect from the matrix. The limit of detection was found to be 0.3-0.8% (depending on the impurity) corresponding to 1.9-5.1 ng. The limit of quantification was found to be 0.8-2.5% (depending on the impurity) corresponding to 5.2-16 ng. The recovery was found to be between 90 and 101% for the FF-MAS, and 100-129% for the six known impurities. The tested range for FF-MAS was from 320 to 960 ng corresponding to 50-150% of the nominal concentration (640 ng, injection volume is 100 microl). The linearity of each compound (FF-MAS and the six impurities) was investigated. The squared correlation coefficient (r(2)) was 0.999 for FF-MAS (50-150% level) and 0.977-0.998 for the six known impurities (at four levels: 0.20, 0.50, 1.00, 2.00%). The R.S.D. in the repeatability study was found to be 9.2% for the total amount of impurities, and 10.4% for single impurities. The R.S.D. in the intermediate precision study was found to be 10.9% for total impurities, and 12.0% for single impurities. The validation results showed that the method was suitable for the purity analysis. The validated method was then ready for use for samples analysis of phase II clinical studies and the stability investigations of the pharmaceutical product.     

On the organic solvent and thermostability of the biocatalytic redox system of Rhodococcus ruber DSM 44541

The sec-alcohol dehydrogenase activity of whole cells of Rhodococcus ruber DSM 44541 has been employed as an efficient biocatalytic redox system due to the use of acetone and 2-propanol at elevated concentrations for cofactor regeneration in the oxidation and reduction mode, respectively, and external addition of NADH/NAD(+) can be omitted. The operational half-life time of the redox system is 29 hours in 20% v/v acetone and 37 hours in 30% v/v 2-propanol. The Redox system allows the enantioselective oxidation of sec-alcohols and the asymmetric reduction of ketones to furnish (S)-configurated alcohols in high optical purity. The stability of the cells towards further organic solvents was investigated. In addition, the system displays thermostability of up to 60 degrees C and pH stability of up to pH 11. The system represents a simple to handle tool for environmentally benign redox reactions.     

Structural and kinetic analysis of drug resistant mutants of HIV-1 protease.

Mutants of HIV-1 protease that are commonly selected on exposure to different drugs, V82S, G48V, N88D and L90M, showed reduced catalytic activity compared to the wild-type protease on cleavage site peptides, CA-p2, p6pol-PR and PR-RT, critical for viral maturation. Mutant V82S is the least active (2-20% of wild-type protease), mutants N88D, R8Q, and L90M exhibit activities ranging from 20 to 40% and G48V from 50 to 80% of the wild-type activity. In contrast, D30N is variable in its activity on different substrates (10-110% of wild-type), with the PR-RT site being the most affected. Mutants K45I and M46L, usually selected in combination with other mutations, showed activities that are similar to (60-110%) or greater than (110-530%) wild-type, respectively. No direct relationship was observed between catalytic activity, inhibition, and structural stability. The mutants D30N and V82S were similar to wild-type protease in their stability toward urea denaturation, while R8Q, G48V, and L90M showed 1.5 to 2.7-fold decreased stability, and N88D and K45I showed 1.6 to 1.7-fold increased stability. The crystal structures of R8Q, K45I and L90M mutants complexed with a CA-p2 analog inhibitor were determined at 2.0, 1.55 and 1.88 A resolution, respectively, and compared to the wild-type structure. The intersubunit hydrophobic contacts observed in the crystal structures are in good agreement with the relative structural stability of the mutant proteases. All these results suggest that viral resistance does not arise by a single mechanism.     

Field response of the pine sawfly Neodiprion sertifer to the pheromone (2S,3S,7S)-diprionyl acetate and its stereoisomers

All eight optical isomers of 3,7-dimethyl-2-pentadecanyl acetate (diprionyl acetate), of high optical purity (> 97.4%), were tested for a behavioural activity on male pine sawflies, Neodiprion sertifer (Geoffr.) (Hymenoptera: Diprionidae), in northern Europe. Males were strongly attracted to (2S,3S,7S)-diprionyl acetate. Addition of more than 0.1% of the (2S,3R,7R)-isomer reduced the catch and above 2% the attraction was completely inhibited. Contrary to what has been reported for North American and Japanese populations, so significant synergistic effect of small amounts of the (2S,3R,7R)-isomer could be demonstrated. The effects of addition of the other six optical isomers alone or in combinations, were also studied, but none was found to be a synergist. The (2S,3R,7S)-isomer had a weak inhibitory effect, and completely inhibited the attraction to the (2S,3S,7S)-isomer when applied in about equal amounts as the attractant. In some cases a reduction in catch was noted when other isomers were tested, but this could be attributed to the very small amounts of the inhibitory (2S,3R,7R)-isomer present in these isomers.     

Purification and Properties of Urease Derived from Hydrated Seeds of Jack Bean, Canavalia ensiformis (L) DC

Urease from jack bean meal and hydrated seeds has been obtained in 25 to 33% yield with specific activity in the range of 1000 to 1070 units/mg protein. A purification of 100 to 130-fold was achieved from meal and fully soaked seeds. Use of β-mercaptoethanol and EDTA was found essential to obtain this high yield and purity. Amino acid analysis showed all 18 amino acids commonly found in proteins. Electrophoresis of urease from soaked seeds (specific activity: 1025 units/mg protein) on a starch-gel block showed 2 peaks. Upon ultracentrifugation of urease samples having a low specific activity (less than 25% pure), the major portion of the urease was probably present in a peak having a sedimentation value of 11 to 12. With relatively pure samples (55-100% pure). S values in the range of 18 to 20 and 24 to 26 were obtained. Usually the purest samples of urease tested without any prior storage lacked the 24 to 26 S peak or the higher polymeric forms. The percentage areas under none of the ultracentrifuge peaks corresponded to the percentage purity of the sample analyzed. It is argued that the physical state of urease in the cell when associated with other seed proteins is as yet uncertain. In crude extracts, a portion of urease exists in a 12 S form but so far data on its origin and specific activity in relation to other species of urease are not available.