人Leptin基因在大肠杆菌中的高效表达、纯化及其生物学活性研究

将人Leptin表达质粒pBV220-OB转化E.coliJM109,经热诱导获得了目的蛋白的表达。经SDS-PAGE鉴定分析,表达产物以包涵体形式存在,目的蛋白表达量占菌体总蛋白的40%以上。通过包涵体分离,Sephacryl S200HR凝胶和DEAE52离子交换层析及Hypersil C18柱反相色谱纯化,获得纯度在95%以上,内毒素含量小于10EU/mg的高纯度的重组人Leptin。Western-blot鉴定表明,纯化表达产物能和抗Leptin抗体特异性结合;蛋白质N端15个氨基酸序列分析结果和预期的序列一致。纯化产物经复性处理,其分子中Cys96和Cys146形成二硫键。体内活性检测显示,纯化和复性的rh-Leptin明显抑制BALB/c小鼠的进食和体重增长,提示其具有明显的生物学活性。     

大黄素升高豚鼠结肠带细胞[Ca~(2 )]_i的特征和GDP的抑制作用

利用Fluo -3荧光探针检测细胞内自由Ca2 浓度([Ca2 ]i),研究了大黄素升高豚鼠结肠带细胞[Ca2 ]i 的量—效关系和动态变化特征,及GDP和胞外Ca2 浓度对其的影响。较低浓度大黄素随药物浓度增加使[Ca2 ]i 显著升高 ,更高浓度大黄素有超最大抑制效应。GDP对大黄素升高细胞[Ca2 ]i 的抑制作用随其浓度增加而增强。GDP和胞外Ca2 浓度影响大黄素诱发的[Ca2 ]i 动态变化的结果表明 :GDP使[Ca2 ]i 峰消失 ,胞外无Ca2 导致[Ca2 ]i 随时间显著下降 ,大黄素升高[Ca2 ]i 作用趋向消失。     

大肠杆菌编码区碱基片段的分析研究

对大肠杆菌1231个编码开始区域和1307个编码终止区域内的6碱基片段、4碱基片段和3碱基片段进行了统计,发现绝大多数4碱基和3碱基模式出现在盯对频率小于2的范围之内;在这两个区域中,出现最多的碱基片段是多聚A;编码开始区域和编码终止区域的碱基构成模式有明显区别;编码开始区域里GGA类的稀有片段恰恰是SD区域最偏好的碱基片段;以TAG或CTA构造的3碱基模式为编码开始区和编码终止区的禁用模式。     

外源蛋白在大肠杆菌中的表达定位策略

外源基因在大肠杆菌中表达是对基因重组技术的成功应用。外源基因在不同的大肠杆菌表达系统中表达产物可能定位于大肠杆菌空间结构的不同位置:胞质,胞质膜,胞周质,胞外膜和胞外培养基,五种表达定位方式各有其特点和用途 。     

利用两种不相容质粒在大肠杆菌中共表达DFF45和DFF40

DNA断裂因子（DNA fragmentation factor,DFF）是细胞凋亡过程中起重要作用的蛋白质之一,它由分子量为45kD和40kD的两个亚基构成,分别称为DFF45和DFF40。利用RT－PCR技术从人宫颈癌细胞系HeLa的总RNA中扩增了DFF45和DFF40的cDNA,分别克隆到到卡那霉素抗性表达载体pET-28a( )中,构建了pET28a-DFF45和pET28a-DFF40。用它们单独转化大肠杆菌BL21（DE3）后,经IPTG诱导都可获得高效表达。表达的重组蛋白质各自约占菌体总蛋白质的56％和22％。再将DFF45的cDNA克隆到氨苄青霉素抗性表达载体pET-21a( )中,得到了pET21a-DFF45。利用二者的不同抗性,将pET28a-DFF40和pET21a-DFF45共同转化大肠杆菌BL21（DE3）,工程菌经IPTG诱导后实现了DFF45和DFF40的共表达,表达产物各约占菌体总蛋白质的30％和17％。为了研究这两种不相容质粒在细菌中共存的稳定性,我们将共转化子在同时含有卡那霉素和氨苄青霉素的液体培养基中连续培养14h,发现此时仍有75％以上的细菌可同时耐受两种抗生素,即同时含有pET28a-DFF45和pET28a-DFF40,说明利用两个具有不同抗性的不相容载体进行蛋白质共表达的方法是可行的。     

Characterization of eukaryotic-like kinase activity in Escherichia coli using the gene-protein database

Abstract The gene-protein database was used to obtain the two-dimensional polyacrylamide gel coordinates of proteins phosphorylated in extracts of Escherichia coli including those phosphorylated by eukaryotic-like kinase activities. These suggest that the phosphoproteins correspond to, or co-migrate with, the product of an open reading frame at 1.3 min (Orf80), Enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (Ptsl), the tRNA synthetase for histidine (HisS), and proteins involved in the response to carbon starvation and quinone treatment.     

pH track of expanding bacterial populations

A method of pH distribution measurements in agar nutrient media containing expanding bacterial populations is described. It is based on measuring pH microsamples taken at different points of the media. The sample volume was 10 microliters. A pH sensitive field effect transistor was used as a measuring electrode. Acidification was found to occur in glucose media, while alkalization occurred in the media containing peptone.     

Mapping of noninvasion TnphoA mutations on the Escherichia coli O18:K1:H7 chromosome

Abstract The most virulent newborn meningitis-associated Escherichia coli are of the serotype O18: K1: H7. We previously isolated a large number of E. coli O18:K1:H7 mutants resulting from transposon Tn phoA mutagenesis that fail to invade brain microvascular endothelial cells. We have now determined the locations of 45 independent insertions. Twelve were localized to the 98 min region, containing a 120 kb segment that is characteristic of E. coli O18:K1:H7. Another, the previously described insertion ibe -10::Tn phoA , was localized to the 87 min region, containing a 20 kb segment found in this E. coli . These noninvasion mutations may define new O18:K1:H7 pathogenicity islands carrying genes for penetration of the blood-brain barrier of newborn mammals.     

Multiple xylanases of Cellulomona fimi are encoded by distinct genes

Cellulomonas fimi genomic DNA encoding xylanase activity has been cloned and expressed in Escherichia coli. As judged by DNA hybridization and restriction analysis, twelve xylanase-positive clones carried a minimum of four different xylanase (xyn) genes. The encoded enzymes were devoid of cellulase activity but three of the four bound to Avicel.