α-银环蛇毒素基因的克隆及其非融合型原核表达研究

根据文献报道α－银环蛇毒素的氨基酸序列推导出其DNA序列,设计并人工合成两两互补的14条寡核苷酸片段。经片段延伸、PCR、克隆,成功构建α-银环蛇毒素基因克隆质粒;质粒经XbaI和EcoRI双酶切回收后连接于表达载体pET28a(+)中,分别转化BL21（DE3）、BL21（DE3）Codonplus、BL21（DE3）plysS进行诱导表达,表达产物经Tris/tricine系统进行SDS-PAGE分析。结果表明：该基因已在大肠杆菌BL21（DE3）宿主菌中进行了非融合表达,其表达量占细菌总蛋白的11.98％,主要以包涵体形式存在;同时对表达条件进行了优化,其表达量可达16.28%。经Westernblot分析,在大约8kDa处出现明显的目的带,与预计蛋白分子量大小一致,说明表达产物与天然α-银环蛇毒素具有相似的免疫原性。表达产物纯化、复性后经动物毒性试验表明：表达的α-银环蛇毒素蛋白具有生物学活性,小鼠腹腔注射其LD50约为1.28μg/g。     

Topiramate reduced sweat secretion and aquaporin-5 expression in sweat glands of mice

Decreased sweat secretion is a primary side effect of topiramate in pediatric patients, but the mechanism underlying this effect remains unclear. This study aimed to better understand how topiramate decreases sweat secretion by examining its effect on the expression of carbonic anhydrase (CA) II and aquaporin-5 (AQP5), total CA activity, as well as on tissue morphology of sweat glands in mice. Both developing and mature mice were treated with a low (20 mg/kg/day) and high dose (80 mg/kg/day) of topiramate for 4 weeks. Sweat secretion was investigated by an established technique of examining mold impressions of hind paws. CA II and AQP5 expression levels were determined by immunofluorescence and immunoblotting and CA activity by a colorimetric assay. In mature mice, topiramate treatment decreased the number of pilocarpine reactive sweat glands from baseline in both the low and high dose groups by 83% and 75%, respectively. A similar decrease was seen in developing mice. Mature mice with reactive sweat glands that declined more than 25% compared to baseline were defined as anhidrotic mice. These mice did not differ from controls in average secretory coil diameter, CA II expression and CA activity. In contrast, anhidrotic mice did show a reduction in membrane AQP5 expression in sweat glands after topiramate delivery. Thus, sweat secretion and membrane AQP5 expression in mouse sweat glands decreased following topiramate administration. These results suggest dysregulation of AQP5 may be involved in topiramate-induced hypohidrosis and topiramate may serve as a novel therapy for hyperhidrosis.     

同源四倍体拟南芥的耐锌性及其机理

锌(Zn)是植物生长发育所必需的微量元素,但其过量积累也会对植物产生毒害作用。通过对同源四倍体拟南芥进行高浓度锌处理,发现四倍体拟南芥对锌具有高耐受性和低积累性的特点。在高浓度锌胁迫条件下,同源四倍体的根长和生物量较野生型对照均有明显增加,并且谷胱甘肽(GSH)的含量也明显升高。定量实时PCR(q RT-PCR)分析表明,一些锌胁迫响应基因的表达水平发生了显著改变。以上结果表明,锌胁迫响应基因表达水平的改变以及GSH含量的升高可能在四倍体拟南芥的耐锌过程中发挥重要作用。     

中老年食蟹猴群中糖尿病相关基因的表达状态

该研究选择10岁以上健康中老年食蟹猴138只,按空腹血糖值(FPG)将其分为低血糖组、血糖正常组和高血糖组。采用全自动血液生化仪分别检测各组血清中总胆固醇(TCHO)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)的水平变化;采用荧光定量PCR分析食蟹猴外周血白细胞中37个糖尿病相关基因的mRNA表达情况。结果表明,血清血脂水平HDL-C、LDL-C、TCHO和TG都与FPG分组无显著相关性(P>0.05),而外周血白细胞中ACE、ACLY、PRKCB1、SLC2A4、SNAP23、VAPA、IGF2BP2、IFNG8个基因的mRNA表达水平随FPG的升高而显著上调(P<0.05)。     

Functional expression, purification, and characterization of human Flt3 ligand in the Pichia pastoris system

Flt3 ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of hematopoietic progenitor and stem cells both in vivo and in vitro. Pichia pastoris transformants secreting high-level rhFL were obtained using 'yeastern blotting' method and the expression level in liquid was about 30 mg/L. rhFL was purified to about 95% purity with overnight dialysis, filtration and an anion-exchange step. Further purification steps employing Sephacryl S-200 and reverse-phase HPLC raised the purity to over 99%. The purified rhFL possessed correct N-terminal amino acid sequence and positive Western blotting bands. SDS-PAGE and mass spectrometry analysis showed molecular weight of rhFL was about 21 and 34 kDa, suggesting that rhFL was glycosylated. The result of capillary electrophoresis showed that its pI is 3.12-4.72. Endo H deglycosylation analysis indicated that there was O-glycosylation besides N-glycosylation in rhFL secreted from P. pastoris. Bioactivity assay showed that the purified rhFL had dose-dependent expansion activity on bone marrow nucleated cells.     

Removal of antibiotic resistance of live vaccine strain Escherichia coli MM-3 and evaluation of the immunogenicity of the new strain

MM-3 was a live vaccine strain candidate for protecting neonatal piglets from diarrhea.Designed in the 1980s,a high degree of protection from colibacillosis was afforded to piglets in a challengestudy and field trials.However MM-3 had a drawback of carrying the antibiotic resistance gene (chloramphenicolacetyltransferase gene,cat).The introduction of a host-plasmid balanced lethal system into the vaccine wasa good idea to solve the problem.The λ-Red recombination system was adopted in this study to realize thereplacement of cat by aspartate-semialdehyde dehydrogenase gene (asd) in the plasmid pMM085.The newplasmid named pMMASD was introduced into an Escherichia coli strain χ6097 and Salmonella typhimuriumχ4072 where the asd gene had been knocked out in their chromosomes.Cultured in an Erlenmeyer flask,expression levels of two antigens K88ac fimbriae and heat-labile enterotoxin B subunit (LTB) in cell lysatewere similar among MM-3,χ4072(pMMASD) and χ6097(pMMASD).However,χ4072(pMMASD) possessedthe more effective secretion mechanism to transport LTB enterotoxin into culture liquid.The relatively higherstability of pMMASD in Salmonella typhimurium χ4072 than that of pMM085 in MM-3 was determined bothin vitro in the absence of selective pressure,and in vivo following oral inoculation.Oral immunization ofBALB/c mice with χ4072(pMMASD) or χ6097(pMMASD) was sufficient to elicit IgA responses in mucosaltissues as well as systemic IgG antibody responses to the K88 fimbriae,while MM-3 failed to elicit specificantibody responses to K88 fimbriae in mucosal tissues.Among three live strains,only χ4072(pMMASD)could develop strong humoral responses against LTB enterotoxin.The results suggest that χ4072(pMMASD)is expected to be a promising live vaccine strain.     

持续电刺激家兔包钦格复合体膈神经放电的观察

目的观察长时持续电刺激家兔Btzinger复合体(Bt.C)对膈神经放电的影响。方法采用10～50μA,40～100Hz,波宽0.3ms的刺激参数,对30只家兔Bt.C进行15～30s的刺激。结果刺激期间,出现膈神经放电的抑制或“切断”,即吸气相减弱或消失;随刺激强度和频率的增加,膈神经放电被抑制的程度逐渐增强;刺激Bt.C所致的吸气抑制存在“习惯化”现象,表现为一定刺激范围内,随刺激时间的延长,膈神经放电积分幅度呈“阶梯”状逐步上升;停止刺激后出现膈神经放电的“反弹”,主要表现为放电幅度明显升高,而后逐渐恢复正常;膈神经放电的“习惯化”具有一定的短时记忆效应。结论非联合性学习和记忆形式存在于Bt.C对呼吸的调节中,与Bt.C有关的神经元突触在功能上具有一定的可塑性。