Multiple induced mutagenesis for improvement of ethanol production by <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Kluyveromyces marxianus GX-15 was mutated multiple times by alternately treatment with UV irradiation and NTG for two cycles. Four mutant strains with improved ethanol yield were obtained. The maximum ethanol concentration, ethanol yield coefficient and theoretical ethanol yield of the best mutant strain, GX-UN120, was 69 g/l, 0.46 g/g and 91%, respectively, when fermenting 150 g glucose/l at 40°C. The corresponding values for GX-15 were 58 g/l, 0.39 g/g and 76%, respectively. GX-UN120 grew well in 11% (v/v) of ethanol, while GX-15 could not grow when ethanol was greater than 8% (v/v).     

Chemiluminescent immunoassay of thyroxine enhanced by microchip electrophoresis

A homogeneous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophoresis separation has been developed. The method deployed the competitive immunoreaction of T4 and horseradish peroxidase (HRP)-labeled T4 (HRP-T4) with anti-T4 mouse monoclonal antibody (Ab). HRP-T4 and the HRP-T4-Ab complex were separated and quantified by using microchip electrophoresis (MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol-H₂O₂ reaction. Due to the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. Under the selected assay conditions, the MCE separation was accomplished within 60 s. The linear range for T4 was 5-250 nM with a detection limit of 2.2 nM (signal/noise ratio = 3). The current method was successfully applied for the quantification of T4 in human serum samples. It was demonstrated that the current MCE-CL-enhanced competitive immunoassay was quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of T4 to assist in the diagnosis of thyroid gland functions.     

表达SLTEC保护性抗原的重组猪霍乱沙门氏菌C500株的构建及生物学特性

摘要:【目的】 利用平衡致死系统构建表达产类志贺氏毒素大肠杆菌(Shiga-like toxin Escherichia coli , SLTEC)保护性抗原的减毒猪霍乱沙门氏菌。【方法】 构建表达SLT-IIeB－FedF的重组质粒 ,再将其电转入终宿主菌减毒猪霍乱沙门氏菌ΔasdC500株中构建成口服活疫苗株 ,经聚丙烯酰胺凝胶电泳检测SLT-IIeB－FedF融合蛋白的表达情况,并观察重组菌体外培养的稳定性。【结果】 　利用宿主-载体平衡致死系统构建了表达SLTEC保护性抗原的重组减毒猪霍乱沙门氏菌     

野化培训大熊猫采食和人为砍伐对拐棍竹无性系种群更新的影响

亚高山竹类占据着野生大熊猫(Ailuropoda melanoleuca)食物组分的99%,竹子的生命周期与大熊猫的生活史密切相关,竹子的更新和生长直接影响着大熊猫的生存与保护.为了弄清大熊猫的采食利用和人为砍伐是否促进或制约竹子的繁殖更新,应用样方法、定位观察法,连续研究了卧龙自然保护区大熊猫栖息地内野化培训大熊猫采食、人为砍伐和对照样方中拐棍竹无性系种群的更新动态.结果表明,在相同种群数量和环境条件的基础上(p>0.05),被大熊猫采食竹子的比例为67.07%,致死率29.07%;人为砍伐竹子的比例为65.67%,致死率46.68%,可见与大熊猫采食相比,砍伐更为影响拐棍竹种群的生存.从出笋数量来看,不同处理方式有利于拐棍竹无性系种群的更新(p<0.05),尤其是人为砍伐措施大大提高了竹子的出笋率,但人为砍伐样方的竹笋质量(地径和株高生长)却远低于大熊猫采食和对照样方,未能达到大熊猫觅食的选择利用标准.不同年份之间,各种处理方式下的拐棍竹出笋数量波动较大(p=0.006～0.035),并随着恢复时间的延长,逐渐趋于相似(2007,p=0 825).不同处理方式之间,拐棍竹无性系种群的年补充率,各年份均具有显著性差异(p<0.05,除2007年外),年死亡率仅2003年和2004年有明显的差异(p<0.05),2005～2007年均不显著(p>0.05).拐棍竹无性系种群的年补充率与年死亡率之间表现出年补充率﹥年死亡率的格局(p<0.05),唯有大熊猫采食样方的2004年和2005年、人为砍伐和对照样方的2005年的年死亡率略高于年补充率(p>0.05),这表明拐棍竹无性系种群对大熊猫采食和人为砍伐具有无性系整合的补偿效应.     

遗传学综合实验的教学改革实践

利用果蝇杂交实验,进行了教学改革的尝试。通过教学实践力求将几个独立的果蝇实验集中于一个,以充分利用实验时间,增加内容,改进方法。在改革的尝试中,学生自主设计实验方案,独立完成实验内容,处于主导地位。充分发挥了学生主动性,达到了培养学生独立思考、实际操作及科研素质等综合能力的目的。     

Identification of three protein disulfide isomerase members from Haemaphysalis longicornis tick

Three genes encoding putative protein disulfide isomerase (PDI) were isolated from the Haemaphysalis longicornis EST database and designed as HlPDI-1, HlPDI-2, and HlPDI-3. All three PDI genes contain two typical PDI active sites CXXC and encode putative 435, 499, and 488 amino acids, respectively. The recombinant proteins expressed in Escherichia coli all show PDI activities, and the activities were inhibited by a PDI-specific inhibitor, zinc bacitracin. Western blot analysis and real-time PCR revealed that three HlPDIs were present in all the developmental stages of the tick as well as in the midgut, salivary glands, ovary, hemolymph, and fatbody of adult female ticks, but the three genes were expressed at the highest level in the egg stage. HlPDI-1 is expressed primarily in the ovary and secondarily in the salivary glands. HlPDI-2 and HlPDI-3 are expressed primarily in the salivary gland, suggesting that the PDI genes are important for tick biology, especially for egg development, and that they play distinct roles in different tissues. Blood feeding induced significantly increased expression of HlPDI-1 and HlPDI-3 in both partially fed nymphs and adults. Babesia gibsoni-infected larval ticks expressed HlPDI-1 and HlPDI-3 2.0 and 4.0 times higher than uninfected normal larval ticks, respectively. The results indicate that HlPDI-1 and HlPDI-3 might be involved in tick blood feeding and Babesia parasite infection in ticks.     

Enantiospecific adjuvant activity of cationic lipid DOTAP in cancer vaccine

Commercially available DOTAP is a racemic mixture of two enantiomers. The adjuvanticity of each isomer was examined using a peptide/lipid complex as a therapeutic vaccine in an established murine cervical cancer model. This simple vaccine consists of a cationic lipid (DOTAP) and a major histocompatibility complex (MHC) class I–restricted epitope of the Human Papillomavirus (HPV) 16 protein E7. Dose-dependent tumor regression experiments have been completed for racemic DOTAP/E7, (R)-DOTAP/E7 and (S)-DOTAP/E7. Tumor-bearing mice treated with (R)-DOTAP/E7 complexes have shown tumor regression in a dose-dependent manner comparable to those mice treated with a racemic DOTAP with E7 peptide. These data are supported by IFN-γ production by CD8⁺ splenocytes, in vivo cytotoxic T-lymphocytes (CTL) response, CD8⁺ tumor-infiltrating lymphocytes (TIL), and IFN-γ production by CD8⁺ TIL in (R)-DOTAP/E7-vaccinated mice. When (S)-DOTAP/E7 is delivered, tumor progression is delayed. While IFN-γ production is absent from CD8⁺ splenocytes in mice vaccinated with (S)-DOTAP/E7, IFN-γ production by CD8⁺ TIL is present, supporting our hypothesis that (S)-DOTAP has limited activity. Activation of bone marrow-derived dendritic cells by the enantiomeric formulations has also been evaluated, as well as cytokine production and toxicity with no considerable differences between the groups. The results show the DOTAP enantiomers act differently as adjuvants in vivo, with (R)-DOTAP being more effective at stimulating a CD8⁺ anti-tumor response.