牛产肠毒素性大肠埃希菌菌蜕的制备及裂解条件的优化

目的制备牛产肠毒素性大肠埃希菌菌蜕,并对裂解条件进行优化。方法将裂解质粒pHH43电转入牛肠产毒素性大肠埃希菌中,37℃培养至A_(600)值为0.6时,升温至42℃诱导裂解,制备菌蜕。同时对起始诱导A_(600)值(0.4、0.6、0.8、1.0、1.2)和培养基成分含量(2/3、3/4、4/5和正常含量)进行优化,并计算裂解效率。结果在起始诱导A_(600)值为0.6,培养基营养成分为正常含量的3/4时,裂解效率最高,达99.93%。结论成功制备了牛产肠毒素性大肠埃希菌菌蜕,并对其制备条件进行了优化,为进一步开展对其免疫效果和佐剂效应等研究奠定了基础。     

2005和2006年流感裂解疫苗接种人体后的反应及免疫效果

目的观察2005、2006年变更流感毒株后制备的裂解疫苗接种人体后反应及免疫效果。方法变更毒株的流感裂解疫苗于2005和2006年,分别在江西九江和河南漯河进行了接种人体后反应及免疫效果观察。结果2年的观察结果显示,生产的流感裂解疫苗接种人体后全身、局部反应轻微,并有较好的抗体应答,对有流感抗体者接种流感疫苗后,仍能促使机体产生高效抗体。结论用变更毒株生产的流感病毒裂解疫苗接种人体后反应和免疫效果均良好。     

细菌微生物对工业油田生产的危害及机理研究

细菌微生物在工业循环冷却水系统及油田回注水系统中大量繁殖,严重影响了正常生产运行。细菌微生物主要包括三类:硫酸盐还原菌、腐生菌及铁细菌。详细论述了细菌微生物对工业油田的危害,并阐述了三种细菌各自的腐蚀作用机理。     

菌影作为疫苗佐剂的潜力及其机制的研究进展

细菌菌影(bacterial ghosts,BGs)是革兰阴性菌的细菌空壳,含有天然的免疫刺激剂(如脂多糖、鞭毛蛋白等),是固有免疫和适应性免疫细胞的强效激活剂。体内外试验研究表明,BGs具有免疫佐剂效应,能够诱导多种细胞产生前炎症细胞因子,后者可招募T细胞和B细胞聚集淋巴结,进而激活一系列体液免疫、细胞免疫及黏膜免疫应答。本文总结了菌影作为疫苗佐剂的广泛应用及其诱导免疫应答的机制,以期为新型疫苗制剂的研制提供思路。     

红茶菌制备细菌纤维素的研究

寻找高效的生产菌和发酵工艺以及廉价高效的培养基是解决当前细菌纤维素产业化面临的高生产成本和低产率等瓶颈问题的重要手段。以红茶菌和木葡糖酸醋杆菌作为生产菌株,比较研究了不同碳源、氮源以及茶叶浓度对两种菌合成细菌纤维素的影响。结果表明,以红茶菌制备的细菌纤维素与木葡糖酸醋杆菌无本质区别;红茶菌生产细菌纤维素的效率显著高于木葡糖酸醋杆菌,产量可提高3倍以上。     

猪胸膜肺炎放线杆菌血清1和5型交叉免疫保护研究

目的探讨猪胸膜肺炎放线杆菌(APP)不同血清型间交叉免疫保护的关系。方法以1型和5型APP参考菌株为实验对象,分别测定半数致死量(LD50),制备灭活细菌抗原和活菌抗原,免疫昆明小鼠,检测ELISA抗体水平及交叉免疫反应,并分别以10LD501型和5型APP菌进行攻击,观察小鼠保护力。结果1型和5型APP菌对小鼠的半数致死量分别为8.0×105.3和5.1×103.83CFU/LD50,死菌抗原和活菌抗原免疫小鼠均可产生高滴度抗体,且1型与5型间存在较强的交叉免疫反应。死菌抗原和活菌抗原免疫小鼠均能对本型APP菌攻击产生保护,保护率在80%以上。1型活菌免疫小鼠对5型菌攻击保护率为70%,5型活菌免疫小鼠对1型菌攻击保护率为90%,1型、5型死菌免疫小鼠对5型、1型菌交叉攻击均无保护作用。结论1型、5型APP活菌和死菌免疫小鼠均可对本型菌攻击产生保护,两型间存在较强的交叉免疫反应,但只有活菌免疫可以产生较好的交叉免疫保护,死菌免疫不能产生交叉免疫保护。     

裂解基因E和核酸酶基因串联表达载体的构建及大肠杆菌菌影的制备

目的构建噬菌体裂解基因E和核酸酶基因串联表达载体,制备高质量的大肠杆菌菌影。方法自行设计一对引物,PCR扩增PhiX174噬菌体裂解基因E,将该基因亚克隆至原核表达载体pGEX-6P-1,构建大肠杆菌菌影的表达载体pGEX-E。在E基因基础上与辅助基因葡萄球菌核酸酶A(SN)基因串联,并插入pGEX-6P-1载体,构建双基因串联高效表达载体pGEX-E-5aaLinker-SN和pGEX-E-15aaLinker-SN,采用CaCl2法将其转入大肠杆菌BL21(DE3),经IPTG诱导,制备大肠杆菌菌影。结果裂解基因E单基因及串联基因已成功插入融合表达载体pGEX-6P-1,所构建的大肠杆菌菌影表达载体pGEX-E、pGEX-E-5aaLinker-SN和pGEX-E-15aaLinker-SN,诱导后经透射电镜及活菌计数显示大肠杆菌已发生不同程度裂解,制成了大肠杆菌菌影。电镜下菌影形态完整,内容物已释放到胞外。结论已成功构建了噬菌体裂解基因E单基因及裂解基因和核酸酶基因串联基因表达载体,并制成了大肠杆菌菌影,为进一步研究菌影这一新型的疫苗及佐剂形式奠定了基础。     

裂解碳九加氢利用技术进展

系统总结了裂解碳九(C9)来源、组成及其综合利用情况;指出裂解碳九加氢技术是裂解碳九综合利用的核心和关键技术;详述了裂解碳九加氢生产高品质芳烃溶荆油或调和油、裂解碳九闪蒸油加氢生产溶剂油及全加氢裂解碳九加氢轻质化增产BTX;重点分析了裂解碳九加氢工艺和催化剂技术,强调了原料预处理、反应工艺和催化剂设计等要点.     

硫酸盐还原菌对20#钢腐蚀行为的影响

为了研究油气田现场生产工况下硫酸盐还原菌对20#钢腐蚀行为的影响,本文通过细菌培养实验、腐蚀失重实验等实验方法,利用激光共聚焦显微镜,扫描电镜等仪器,研究了矿化度、温度、H_2S、CO_2对硫酸盐还原菌(SRB)生长活性及对20#钢腐蚀行为的影响。结果表明:矿化度升高影响SRB细菌活性,SRB细菌导致的腐蚀行为减弱;温度变化对SRB细菌生长活性影响较大,高温下腐蚀主要由溶液自身环境造成;H_2S分压、CO_2分压对SRB细菌生长影响不大。     

喷鼻三价流感裂解疫苗在小鼠体内的免疫原性及免疫保护性

目的 评价喷鼻三价流感裂解疫苗在小鼠体内产生的免疫原性及免疫保护性。方法 选用H1N1、H3N2、B型3种裂解抗原联合后,与佐剂壳聚糖季铵盐(HTCC)水凝胶等体积混合,制备三价流感裂解疫苗。经喷鼻免疫BALB/c小鼠,免疫剂量为每种单价抗原2μg/只,第0及14天各免疫1次,于末次免疫后第14天制备小鼠血清及鼻、肺、阴道灌洗液。血凝抑制法检测小鼠血清的血凝抑制抗体(HI)效价;ELISA法检测小鼠血清中Ig G及其亚型抗体效价和小鼠鼻、肺、阴道灌洗液中sIgA效价;末次免疫14 d,用H1N1流感病毒进行攻毒,观察小鼠体重变化及死亡率。结果 经喷鼻免疫三价流感裂解疫苗的小鼠血清可产生较高效价的HI抗体,IgG抗体亚型以IgG1与IgG2a为主,小鼠鼻、肺、阴道灌洗液中均可检测到特异性sIgA。攻毒后小鼠体重变化较小,14 d后全部存活。结论 经喷鼻免疫三价流感裂解疫苗可激发小鼠产生体液、细胞及黏膜免疫反应,具有良好的免疫保护性。     

Removal of lipid from intact erythrocytes and ghosts by aqueous solutions and its relevance to membrane structure

Molar values for cholesterol, total phospholipid, and individual phospholipid classes of intact erythrocytes and their membranes (ghosts) washed with various aqueous solutions are presented. The data show that lipid can be washed from erythrocyte ghosts prepared rapidly from freshly drawn blood but that lipid is not removed from intact erythrocytes under the same conditions. Thus, it appears that the polar groups of lipids of intact cells are not exposed as they are in ghosts. In the preparation of hemoglobin-free ghosts, up to 25% cholesterol and phospholipid can be removed, while loss of ca. 50% cholesterol and phospholipid from ghosts can be achieved with aqueous solutions containing ethylenediamine tetraacetate. No significant loss of membrane protein was encountered even when almost half of the lipid had been removed from the ghosts. Phospholipid classes were removed to different extents with different wash solutions. Lipid loss from ghosts can be prevented, in part, by adding 0.5% albumin or calcium to wash solutions containing ethylenediamine tetraacetate. These findings contrast a report where insignificant lipid loss was noted in the preparation of hemoglobin-free human erythrocyte membranes, but agree with results reported for bovine red cell ghosts.     

Dendrosomes as novel gene porters‐III

BACKGROUND: It was previously reported that dendrosomes, i.e. neutral, biodegradable, covalent or self‐assembled, hyperbranched, spheroidal nano‐particles with a size ranging from 15 to 100 nm, provide a convenient and efficient means of gene delivery into various kinds of cells such as human hepatoma and kidney cells as well as animal models. RESULTS: New studies via circular dichroism show that hydrophilic and amphipathic dendrosomes either do not affect the DNA structure or moderately transform it from B‐ to A‐conformation. Gene delivery into human liver, kidney, and endothelial cells as well as other animal cells like Bowes, U‐937, Raw, CCRF‐CEM, MOLT‐4, K562, Huh‐7 and VERO reveal that the genes are efficiently expressed and in comparison with other gene porters like Lipofectin or bacterial ghosts, do quite well. It is also shown that dendrosomes are able to deliver genes into cells like endothelials that are usually hard to transfect. Cell culture experiments as well as intraperitoneal/intradermal injections of dendrosomes into mice establish their nontoxicity (up to 2.5 mg kg^?1 of animal weight in the latter case). Studies on immunization of BALB/c mice using conventional adjuvants such as aluminium phosphate, C_pG motif and one of the dendrosomes, indicate that the latter leads to the mildest initial response development while exceeding them afterwards. CONCLUSION: CD studies reveal that, owing to the neutrality of dendrosomes, formation of Den/DNA complexes is accompanied by slight structural modifications of DNA cell culture, and animal studies reveal that dendrosomes are inert, non‐toxic and highly efficient gene porters that perform at extremely low doses. In comparison with bacterial ghosts and some common porters, they are efficient in delivery of genes into animals and a variety of cells including those that are usually hard to transfect. Copyright © 2008 Society of Chemical Industry     

Periodate-induced lipid oxidation of erythrocyte membranes

Exposure of human erythrocyte ghosts to 0.2–5 mM periodate at 0 C for 15 min resulted in an increase of thiobarbituric acid-reactive substances and fluorescent materials in the membranes. This increase was suppressed by radical scavengers, butylated hydroxytoluene and thiourea, indicating that periodate caused lipid oxidation of ghosts. A role of hemoglobin in the periodate-induced lipid oxidation of ghosts was suggested by the fact that the oxidation was augmented by hemoglobin and inhibited by an iron chelator, desferrioxamine. Treatment of ghosts with periodate caused membrane protein cross-linking with and without disulfide bridge. Erythrocytes were also susceptible to lipid oxidation by periodate, but only disulfide-mediated protein cross-linking was observed. Erythrocytes treated with neuraminidase or trypsin were less susceptible, but those treated with neuraminidase along with galactose oxidase were nearly as susceptible as untreated cells. The effect of galactose oxidase was diminished by reduction of the enzyme-treated cells with borohydride. These results indicate that the aldehyde moieties generated by periodate at the sialyl residues or those generated by galactose oxidase at the terminal galactosyl orN-acetyl galactosaminyl residues of the membrane glycoconjugates play a stimulating role in the periodate-induced membrane lipid oxidation.     

Autoxidation and effects of pro- and antioxidants in lyophilized red blood cell membranes

Oxygen uptake and effects of pro- and antioxidants have been compared at 80 C in lyophilized red blood cell (RBC) bilayer membranes (ghosts). In this dry and relatively immobile system, catalytic metals have pronounced effects. When ghosts are prepared in the usual manner, in phosphate buffer, hypotonic saline, or deionized water (DI), oxygen uptake is extremely slow and limited unless: (a) catalytic metal, e.g., cobaltous ion, is supplied in the absence of metal-complexing buffers and (b) residual phosphate buffer is removed by repeated deionized water or hypotonic saline washes. Ghosts adsorb d-α-tocopherol strongly from 1% alcohol emulsion in buffer in amounts far above normal RBC concentrations, whereas synthetic antioxidant uptake seldom exceeds the normal tocopherol level. Uptake and effectiveness of antioxidants introduced by either perfusion or in the vapor phase, and resistance of ghosts to autoxidation are discussed as they relate to protection of lyophilized membranes and freeze-dried foods.     

Semiconductor-electrochemical aspects of bacterial leaching. I. Oxidation of metal sulphides with large energy gaps

Thiobacillus ferrooxidans has been cultivated successfully on synthetic metal sulphides with large energy gaps (CdS, ZnS) as the only energy source for many culture generations during a period of 4 years. The results obtained, which were quantitatively evaluated by calculations of electron transfer probabilities, show that a direct electron transfer from the metal sulphide valence band to the bacterial metabolic system (hole injection into the valence band of the sulphide) has to be excluded as the cause for the enhanced oxidative dissolution for energetic reasons. A detailed analysis of the dynamics of the metal sulphide aqueous electrolyte interface reveals that protons are involved in the mechanism on which bacterial activity is based. By reacting chemically with the metal sulphide surface they break chemical bonds and shift electronic states energetically into the forbidden energy gap to produce surface states which can be chemically described as ?SH? groups. These control the rate of dissolution of the metal sulphide and are removed by bacterial activity. In this way the proton is recycled and its action can be considered catalytic. For sulphides in which the valence band of the semiconductor is derived from metal orbitals instead of from sulphur orbitals, this mechanism is bound to fail. MoS₂ and WS₂ are discussed as examples of such metal sulphides which are not a suitable energy source for bacteria. Some kinetic aspects of the bacterial surface reaction are discussed.     

Lipidic characterization of full-grown amphibian oocytes and their plasma membrane-enriched fractions

The content and composition of neutral lipids and phosphoglycerides from full-grown prophase-arrestedBufo arenarum Hensel oocytes and from their ghost preparations were studied. The ghosts obtained are highly enriched in plasma membrane as suggested by the activity of 5′-nucleotidase, a marker enzyme, and the level of typical membrane components such as sphingomyelin, phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidic acid. In whole oocytes, triacylglyceride (TAG) comprises about 60% of the total lipids followed by phosphatidylcholine (PC), cholesterol, and phosphatidylethanolamine (PE). TAG and diacylglycerides have a similar unsaturation index. PC and PE account for about 80% of the phosphoglycerides in the whole oocyte and in their plasma membrane-enriched fractions. Arachidonic acid (20∶4n−6), 18∶0, and 16∶0 make up about 80 mol% of the total fatty acids in Pl in whole oocytes and ghost fractions. The unsaturation index in PS is higher in intact oocytes than in ghost preparations, probably owing to the significant amount of 20∶4n−6 which comprises 23 mol% of the total fatty acids in whole oocytes. The fatty acid profile in phosphatidic acid from whole oocytes is rather different from that in ghosts. Sphingomyelin contains mainly saturated and monounsaturated fatty acids, 24∶1 being the principal very long chain unsaturated fatty acid in both oocytes and ghosts.     

Solubility of bacterial cellulose and its structural properties

Based on experiments conducted, it has been found that bacterial cellulose, like spruce cellulose, is soluble in an aqueous NaOH solution with the concentration of 8.5% at a temperature of −5°C if the polymerization degree of the cellulose does not exceed 400. When 1% of urea is added to the NaOH solution, the solubility of cellulose increases; and, in this solvent, bacterial cellulose may be dissolved so long as its polymerization degree is below 560. The results of these experiments are of great practical importance since they point to the possibility of the preparation of cellulose spinning solutions suitable for fiber formation. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 67:1871–1876, 1998