冻干人用狂犬病疫苗(Vero细胞)稳定剂配方的筛选

目的筛选出适合冻干人用狂犬病疫苗(Vero细胞)的热稳定性好、无明胶的稳定剂配方。方法以水分、外观、效价、热稳定性效价为指标,尤其是效价和热稳定性效价,对A、B、C、D、E、F、G等6种稳定剂配方进行系统的优化组合筛选,6种稳定剂配方分别含有蔗糖、乳糖、人血白蛋白、甘氨酸、精氨酸、明胶、尿素、甘露醇、右旋糖苷,其中A为现行疫苗生产配方。运用单因素五元设计法,筛选出最优稳定剂组合。结果方差分析结果显示D组配方对冻干人用狂犬病疫苗(Vero细胞)的M-ABT结果 13.95,37℃放置4周M-ABT结果 13.05;NIH效价1.94,37℃放置4周NIH效价1.56。结论 D组配方对冻干人用狂犬病疫苗(Vero细胞)有较好的保护作用。     

水痘疫苗新型疫苗冻干保护剂的研究

目的:开发出新型号水痘疫苗保护剂替代现行含明胶、人血白蛋白疫苗保护剂,提高病毒保护效果及疫苗受种人群使用安全性。方法:(1)选取右旋糖酐、乳糖、甘露醇等辅料按不同配方进行制备疫苗保护剂。(2)使用不同配方制备的疫苗保护剂,应用细胞工厂水痘疫苗制备工艺进行生产水痘疫苗。(3)使用不同配方制备的疫苗保护剂生产的水痘疫苗与现行疫苗保护剂生产的水痘疫苗进行对比。结果:对比试验结果显示,使用右旋糖酐、乳糖、甘露醇等制备的疫苗保护剂,与现行疫苗保护剂比较无差异。结论:使用右旋糖酐、乳糖、甘露醇等制备的疫苗保护剂比较现行使用的疫苗保护剂,由于不含明胶及人血白蛋白,因此使该疫苗冻干保护剂的安全性、可靠性有所提高,并且疫苗冻干保护剂的成本将有所下降。     

无明胶布氏菌活疫苗冻干稳定剂的筛选

目的为皮上划痕人用布氏菌活疫苗筛选存活率高、无明胶冻干稳定剂。方法以冻干活菌存活率为指标,对甘油、甘露醇、蔗糖、葡萄糖、乳糖、谷氨酸钠、甘氨酸、谷氨酸、脯氨酸和硫脲等10种稳定剂通过单因素筛选法,筛选出冻干存活率高的4种单因素稳定剂成分;将4种单因素稳定剂成分进行正交试验优化,筛选出最优稳定剂组合。结果单因素试验结果显示,甘油、葡萄糖、谷氨酸钠和硫脲4种稳定剂成分冻干后活菌存活率较高,对布氏菌活疫苗具有良好保护效果。通过正交试验筛选出最优稳定剂配方中四组分的质量分数分别为甘油1.5%、葡萄糖5%、硫脲1.5%、谷氨酸钠1.0%,该配方的冻干存活率可达81.5%。结论无明胶冻干稳定剂对布氏菌活疫苗具有较好的保护作用。     

基于多种微生物的毒性检测MTOXPlate冻干板制备及优化

[目的]制备基于多种微生物的生物毒性检测MTOXPlate冻干板,从而简化操作程序,使其能更灵敏、方便、快速地进行生物毒性测试.[方法]采用真空冷冻干燥法将指示微生物固定在微孔板中.以存活率为指标,利用荧光分光光度法确定冻干保护剂的组成.通过分析毒性响应灵敏度对检测条件进行优化.[结果]MTOXPlate的冻干保护剂组成为8％海藻糖、2％葡萄糖、2％甘露醇、1％谷氨酸钠.在此组成下,11株菌株的平均存活率最高,可达89.41％.复活介质与测试样品同时加入,在缩短了测试时间的同时也提高了检测灵敏度.该冻干板具有良好的灵敏度和重现性,能较好地满足污染物生物毒性测试的需要.[结论]MTOXPlate冻干板可作为一种新型的生物毒性测试技术,在生态毒理检测领域推广应用.     

木犀草素脂质体冻干工艺优化及其对LX-2细胞的影响

[目的]优化木犀草素脂质体的冻干工艺,考察其对LX-2细胞增殖的影响。[方法]采用冷冻干燥法制备木犀草素脂质体冻干粉,以外观、再分散性、粒径及包封率为指标,通过单因素考察和Box-Behnken响应面法优选最佳冻干工艺,MTT法检测其对LX-2细胞增殖的影响。[结果]最佳冻干工艺为预冻温度-80℃,预冻时间12 h,干燥时间24 h,冻干保护剂为总量8%的蔗糖-乳糖-甘露醇(1.0∶1.0∶2.0)联合使用;与冻干前比较,冻干粉在4℃下30 d内较稳定;脂质体对LX-2细胞的抑制作用强于原药(P<0.01),并呈浓度和时间依赖性。[结论]按最优工艺制备的脂质体冻干粉包封率为89.03%,与预测值90.12%吻合度高达98.79%;且脂质体对LX-2细胞增殖的抑制率显著高于原药(P<0.01)。     

用巨细胞病毒抗原致敏的冻干血球作间接血凝试验

本文报告用巨细胞病毒(CMV)抗原致敏的冻干绵羊红细胞(简称冻干血球)做间接血凝试验。用5％正常兔血清和10％蔗糖磷酸盐缓冲液作保护剂,将巨细胞病毒抗原致敏的绵羊红细胞进行真空冷冻干燥,制成了冻干血球。经反复检测发现,用含百万分之一Tween20、2％正常兔血清的磷酸缓冲液稀释冻干血球,可消除冻干过程中产生的非特异性凝集反应。所建立的方法重复性较好,特异性与ELISA相同,敏感性比CF高。     

冻干高活力纳豆芽胞杆菌菌粉保护剂的筛选和优化

【目的】对冻干高活力纳豆芽胞杆菌菌粉保护剂进行筛选和优化研究,提高菌粉活菌存活率。【方法】采用单因素实验和正交实验设计,通过测定活菌存活率,筛选出最佳保护剂的配方;并研究采用优化后冻干保护剂制备的菌粉在20°C、4°C、25°C下的保存稳定性。【结果】纳豆芽胞杆菌的有效保护剂是:脱脂乳粉、甘露醇、L-抗坏血酸钠。最佳冷冻干燥保护剂配方是:脱脂乳粉10%+甘露醇4%+L-抗坏血酸钠1%,存活率达到91.63%。菌粉在20°C、4°C、25°C下保存12个月后,存活率分别为:88.79%、70.16%和10.52%,说明菌粉在20°C和4°C下保存稳定性较好,25°C下稳定性比较差。【结论】对纳豆芽胞杆菌冻干菌粉保护剂的优化,对纳豆芽胞杆菌的应用、活菌产品的质量稳定及新产品的研发均有一定的指导意义。     

冻干甲型肝炎-腮腺炎联合疫苗猴体安全性及免疫原性研究试验

为了观察冻干甲型肝炎-腮腺炎联合疫苗免疫恒河猴后的安全性及免疫原性,用静脉注射和丘脑注射的方式接种疫苗,观察恒河猴的临床症状、体征、生化、免疫学反应以及脑和肝组织的病理变化。结果未见临床症状和体征的异常改变,ALT正常,抗-HAV、抗流行性腮腺炎病毒的抗体在观察期内持续阳性,脑组织和肝组织无病毒性肝炎和腮腺炎病毒引起的病理性改变。因此该冻干甲型肝炎堋;腺炎联合疫苗抗原问无干扰,具有良好的安全性及免疫原性。     

花菇的冷冻干燥技术研究

实验研究用板层导热法研究了花菇的冻干特性,获得了新鲜花菇的冻干曲线,分析了花菇冻干过程,测定和比较了新鲜花菇和冻干花菇的营养成份。证实试验机的适应性并确定了花菇的冻干工艺,为工业生产提供了理论依据和参考价值。     

应用生物反应器建立人用冻干狂犬病疫苗(Vero细胞)生产工艺的研究

应用15L生物反应器,采用片状载体对Vero细胞进行高密度培养、制备高毒力滴度的狂犬病毒收获液,经纯化后生产人用冻干狂犬病疫苗。采用15L生物反应器对培养方法(批次培养和连续灌流培养)进行试验,收获高毒力滴度的狂犬病毒收获液并制备人用冻干狂犬病疫苗。结果表明:Vero细胞在接种狂犬病毒后可以连续收获病毒液达到25d以上,冻干狂犬病疫苗的效价可以达到5.54IU/剂。本工艺可以用于进行大规模的人用冻干狂犬疫苗的生产。     

Kinetics and subunit interaction of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system

Purified mannitol-specific enzyme II (EIImtl), in the presence of the detergent Lubrol, catalyzes the phosphorylation of mannitol from P-HPr via a classical ping-pong mechanism involving the participation of a phosphorylated EIImtl intermediate. This intermediate has been demonstrated by using radioactive phosphoenolpyruvate. Upon addition of mannitol, at least 80% of the enzyme-bound phosphoryl groups can be converted to mannitol 1-phosphate. The EIImtl concentration dependence of the exchange reaction indicates that self-association is a prerequisite for catalytic activity. The self-association can be achieved by increasing the EIImtl concentration or at low concentrations of EIImtl by adding HPr or bovine serum albumin. The equilibrium is shifted toward the dissociated form by mannitol 1-phosphate, resulting in a mannitol 1-phosphate induced inhibition. Mannitol does not affect the association state of the enzyme. Both mannitol and mannitol 1-phosphate also act as classical substrate inhibitors. The apparent Ki of each compound, however, is approximately equal to its apparent Km, suggesting that mannitol and mannitol 1-phosphate bind at the same site on EIImtl. Due to strong inhibition provided by mannitol and mannitol 1-phosphate in the exchange reaction, the kinetics of this reaction cannot be used to determine whether the reaction proceeds via a ping-pong or an ordered reaction mechanism.     

Mannitol-specific enzyme II of the bacterial phosphotransferase system. II. Reconstitution of vectorial transphosphorylation in phospholipid vesicles

Purified mannitol Enzyme II from Escherichia coli was reconstituted in phospholipid vesicles employing the octylglucoside dilution procedure and was shown to catalyze vectorial mannitol 1-phosphate:mannitol transphosphorylation. Reconstitution of the enzyme into liposomes showed a marked dependency upon the octylglucoside concentration with an optimum at 1.2%. The reconstituted transphosphorylation activity exhibited an absolute dependence upon mannitol 1-phosphate as the phosphoryl donor, was sensitive to N-ethylmaleimide, and had a pH optimum near 6. The intravesicular radiolabeled mannitol phosphate could be released from the proteoliposomes by the addition of either 50 microM unlabeled mannitol or 0.5% sodium dodecyl sulfate. The rate of formation of intraliposomal mannitol phosphate, measured as a function of the mannitol Enzyme II concentration, showed a sigmoidal response, suggesting that at high enzyme concentrations the mannitol Enzyme II exists in an aggregated or oligomeric state and that this form is more active than the monomeric or dissociated form of the enzyme in catalyzing the vectorial mannitol transphosphorylation reaction.     

Evidence for two distinct conformations of the Escherichia coli mannitol permease that are important for its transport and phosphorylation functions

Column chromatography of the Escherichia coli mannitol permease (mannitol-specific enzyme II of the phosphotransferase system) in the presence of deoxycholate has revealed that the active permease can exist in at least two association states with apparent molecular weights consistent with a monomer and a dimer. The monomeric conformation is favored by the presence of mannitol and by the phosphoenolpyruvate (PEP)-dependent phosphorylation of the protein. The dimer is stabilized by inorganic phosphate (Pi), which also stimulates phospho-exchange between mannitol and mannitol 1-phosphate (a partial reaction in the overall PEP-dependent phosphorylation of mannitol). Kinetic analysis of the phospho-exchange reaction revealed that Pi stimulates phospho-exchange by increasing the Vmax of the reaction. A kinetic model for mannitol permease function is presented involving both conformations of the permease. The monomer (or a less-stable conformation of the dimer) is hypothesized to be involved in the initial mannitol-binding and PEP-dependent phosphorylation steps, while the stably associated dimer is suggested to participate in later steps involving direct phosphotransfer between the permease, mannitol and mannitol 1-phosphate.     

Fluorescence characterization of the low pH-induced change in diphtheria toxin conformation: effect of salt

When injected into rats, a certain amount of mannitol is taken up by the liver and is associated with sedimentable structures. Isopycnic centrifugation in a sucrose gradient shows that a large part of mannitol is present in mitochondria, what remains is located in the lysosomes. The hypotonic release of mannitol present in organelles shows that the polyol is shared between mitochondria and lysosomes. The trapping of mannitol in lysosomes could result from the heterophagic or autophagic function of the lysosomes; the mechanism of its accumulation in mitochondria is still unexplained.     

Mannitol metabolism in the phytopathogenic fungus Alternaria alternata

Mannitol metabolism in fungi is thought to occur through a mannitol cycle first described in 1978. In this cycle, mannitol 1-phosphate 5-dehydrogenase (EC 1.1.1.17) was proposed to reduce fructose 6-phosphate into mannitol 1-phosphate, followed by dephosphorylation by a mannitol 1-phosphatase (EC 3.1.3.22) resulting in inorganic phosphate and mannitol. Mannitol would be converted back to fructose by the enzyme mannitol dehydrogenase (EC 1.1.1.138). Although mannitol 1-phosphate 5-dehydrogenase was proposed as the major biosynthetic enzyme and mannitol dehydrogenase as a degradative enzyme, both enzymes catalyze their respective reverse reactions. To date the cycle has not been confirmed through genetic analysis. We conducted enzyme assays that confirmed the presence of these enzymes in a tobacco isolate of Alternaria alternata. Using a degenerate primer strategy, we isolated the genes encoding the enzymes and used targeted gene disruption to create mutants deficient in mannitol 1-phosphate 5-dehydrogenase, mannitol dehydrogenase, or both. PCR analysis confirmed gene disruption in the mutants, and enzyme assays demonstrated a lack of enzymatic activity for each enzyme. GC-MS experiments showed that a mutant deficient in both enzymes did not produce mannitol. Mutants deficient in mannitol 1-phosphate 5-dehydrogenase or mannitol dehydrogenase alone produced 11.5 and 65.7 %, respectively, of wild type levels. All mutants grew on mannitol as a sole carbon source, however, the double mutant and mutant deficient in mannitol 1-phosphate 5-dehydrogenase grew poorly. Our data demonstrate that mannitol 1-phosphate 5-dehydrogenase and mannitol dehydrogenase are essential enzymes in mannitol metabolism in A. alternata, but do not support mannitol metabolism operating as a cycle.     

Regulation of mannitol biosynthesis and degradation by Cryptococcus neoformans.

Cryptococcus neoformans, an encapsulated yeast that is an opportunistic pathogen of AIDS patients, produced and secreted mannitol when incubated with an appropriate carbon source. Glucose, fructose, and mannose were good growth substrates and were converted to mannitol. Maltose and xylose were good growth substrates but were not converted to mannitol. Cells of C. neoformans that were grown on a non-mannitol-generating carbon source, such as peptone or xylose, were able to convert glucose to mannitol only after a prolonged lag period in the presence of glucose. It was concluded that the enzymes of the mannitol biosynthetic pathway were not constitutively expressed but were induced in response to glucose or to a glucose metabolite. Enzymes required to catabolize mannitol, however, were constitutively expressed. The production of mannitol was inhibited by anaerobiosis, by the respiratory poison rotenone, and by polyethylenesulfonate, a specific inhibitor of fungal NADP-dependent dehydrogenases. When cells were incubated with deuterated glucose, the deuterium content of the mannitol produced was much lower than that of the glucose precursor, indicating that the glucose was diluted by an intracellular pool of an intermediate. We had previously shown that C. neoformans contains a large intracellular pool of glucose 6-phosphate, and we now conclude that this pool of glucose 6-phosphate is metabolically active.     

Membrane Translocation of Mannitol in Escherichia coli Without Phosphorylation

Galactosyl-mannitol can be transported into cells of Escherichia coli by beta-galactoside permease and can be hydrolyzed rapidly to mannitol and galactose by beta-galactosidase. When a mutant strain lacking enzyme I of the phosphoenolpyruvate phosphotransferase system and constitutive in the lactose system was presented with galactosyl-mannitol in which the mannitol moiety was labeled with (3)H, the liberated mannitol remained inside the cell if the Enzyme II complex of the phosphoenolpyruvate phosphotransferase system for mannitol was uninduced. It is postualted that one of the enzyme II proteins can still catalyze translocation of mannitol across the cell membrane even when phsophorylation is not possible.     

Biosynthesis and catabolism of mannitol is developmentally regulated in the protozoan parasite Eimeria tenella

The mannitol cycle is a metabolic branch of the glycolytic pathway found in Eimeria tenella. In this paper, we describe the biosynthesis and consumption of mannitol during parasite development. Low micromolar levels of mannitol were detected in all of the asexual stages and mannitol production increased sharply during the sexual phase of the life cycle. Unsporulated oocysts had high mannitol content (300 mM or 25% of the oocyst mass). Mannitol-1-phosphate dehydrogenase (M1PDH), the first committed step of the mannitol cycle, was also elevated in sexual stages and this coincides with mannitol levels. Approximately 90% of the mannitol present in unsporulated oocysts was consumed in the first 15 hr of sporulation, and levels continued to drop until the sporulation process was complete at approximately 35 hr. Thus, mannitol appears to be the "fuel" for sporulation during the vegetative stage of the parasite life cycle. Evaluation of oocyst extracts from 6 additional Eimeria species for mannitol content and the presence of M1PDH indicated that the mannitol cycle was broadly present in this genus. This finding combined with the lack of mannitol metabolism in higher eukaryotes makes this pathway an attractive chemotherapeutic target.     

Identification of a mannitol transporter, AgMaT1, in celery phloem

A celery petiole phloem cDNA library was constructed and used to identify a cDNA that gives Saccharomyces cerevisiae cells the ability to grow on mannitol and transport radiolabeled mannitol in a manner consistent with a proton symport mechanism. This cDNA was named AgMaT1 (Apium graveolens mannitol transporter 1). The expression profile in source leaves and phloem was in agreement with a role for mannitol in phloem loading in celery. The identification in eukaryotes of a mannitol transporter is important because mannitol is not only a primary photosynthetic product in species such as celery but is also considered a compatible solute and antioxidant implicated in resistance to biotic and abiotic stress.     

The uptake of mannitol by higher plants

Experiments with youngHordeum sativum andHelianthus annus plants showed that in the excretion of mannitol in the guttation liquid observed byGroenewegen andMills (1960) after uptake by the root system of plants, the osmotic concentration of mannitol in the nutrient medium and the temperature are significant. The beginning of mannitol excretion during guttation is accelerated considerably by the increase of the osmotic concentration of mannitol in the nutrient medium and the rising temperature. The osmotic concentration of mannitol is also important for the duration of mannitol excretion in the guttation liquid after transfer of the plants into a nutrient medium without mannitol. In the presence of mannitol in the nutrient medium water uptake by the root system and growth are inhibited and the tissues of the organs above ground and of the root system are dehydrated. The inhibitory effect of mannitol on the water uptake by the root system is immediate.