Population structure and distribution of virulence-related genes of Bacteroides fragilis isolates from Korea and Japan

Sequences for rpoB, gyrB, pdiA, and ompA were determined from 63 Bacteroides fragilis isolates, which were from Korea and Japan and include 4 reference strains. All 4 gene sequences supported clear separation of the cfi⁺ group from the cfi⁻ group. Combined sequences of the 60 division I isolates (cfi⁻) produced 45 different clones. Apparent discordance of gene trees, index of association, maximum likelihood test, and homoplasy ratio all supported a high frequency of recombination. There was no association between the presence of virulence-related genes and phylogenetic clustering in any gene tree.     

猴免疫缺陷病毒外膜糖蛋白基因序列重组和突变克隆的转染研究

目的　研究猴免疫缺陷病毒 (SIV)外膜糖蛋白 (envGP)基因片段与病毒复制功能的关系。方法　利用两病毒株共有的内切酶位点 ,将非致病性SIVmac 142株envGP区的DNA片段与致病性SIVmac 2 39株的对应区域进行置换 ,构成多个重组体。RFLP和部分序列测序确定后 ,用等量突变病毒 (3μg)转染CD4 T淋巴细胞CEM× 174细胞系 ,用ELISA监测培养液中SIV核心蛋白P2 7水平的变化 ,以判定重组病毒的复制能力。结果　与SIVmac 2 39株相比 ,SIVmac 2 39envGP重组体 (SIV mac142 /2 39envGP/142 )仍保持高度的复制活性 ,SIVmac2 39gp41(SIVmac142 /2 39gp41/142 )或SIV mac2 39N gp41(SIVmac142 /2 39N gp41/142 )的复制能力明显降低 ,而SIVmac2 39C gp41(SIVmac142 /2 39C gp41/142 )重组体无复制活性。结论　envGP基因是SIVmac2 39株复制能力或毒力的重要调节因素。     

Recombination analysis based on the HAstV-2 and HAstV-4 complete genomes

Complete genome sequences of previously unstudied human astrovirus subgenotypes – HAstV-2a and HAstV-2c – and two isolates of a rare genotype HAstV-4 have been determined. These isolates were recovered from fecal samples of young children hospitalized with acute intestinal infections in Novosibirsk (Russia). Three of the four sequenced isolates (HAstV-2a, HAstV-2c, and HAstV-4) are recombinants. It has been shown that all known HAstV-2 genomes have emerged via recombination; the HAstV-1 and HAstV-4 genotypes contain both recombinant and non-recombinant isolates; and all HAstV-3, HAstV-5, and HAstV-6 whole-genome sequences display no reliable signs of recombination. The average mutation accumulation rate has been determined based on an extended ORF2 fragment and amounts to 1.0 × 10⁻³ substitutions per site per year. The evolutionary chronology of current HAstV genotypes has been reconstructed.     

抗KDRscFv复制缺陷型腺病毒载体的构建及鉴定

目的构建抗KDRscFv复制缺陷型腺病毒载体，为抗KDRscFv抗肿瘤血管生成的体内应用打下实验基础。方法采用重叠延伸PCR方法构建IL-2信号肽及抗KDRscFv融合基因，克隆于腺病毒穿梭质粒pAdTrack-CMV，转染含pAdEasy-1的BJ5183细菌，经细菌内同源重组产生携带抗KDRscFv基因（含信号肽）的重组腺病毒载体pad-抗 KDRscFv，经脂质体法转染293细胞，包装产生携带抗KDRscFv基因的重组腺病毒Ad-抗 KDRscFv。结果构建了表达抗KDRscFv基因的复制缺陷型腺病毒，且该重组腺病毒能在肝癌细胞株HepG2中高效表达。结论成功构建表达抗KDRscFv基因的复制缺陷型腺病毒载体，为进一步研究抗KDR-scFv在肿瘤治疗中的作用研究提供实验基础。     

日本血吸虫重组两歧双歧杆菌(pGEX-Sj32)疫苗构建及鉴定

目的 构建和鉴定日本血吸虫重组两歧双歧杆菌(Bifidobacterium bifidum,Bb)pGEX-Sj32疫苗.方法 从重庆医科大学附属第一医院传染病寄生虫病研究所构建的重组大肠埃希菌BL21 (pET28α-Sj32)中抽提质粒pET28α-Sj32,通过PCR扩增Sj32抗原编码基因;将Sj32基因定向克隆至大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT中,构建重组质粒pGEX-Sj32,将该重组质粒转化至大肠埃希菌BL21感受态细胞(DE3),抽提质粒后进行双酶切鉴定;采用电穿孔法,将重组质粒pGEX-Sj32转化至Bb,构建Bb(pGEX-Sj32)疫苗,再从该疫苗中抽提重组质粒pGEX-Sj32,以其为模板进行PCR鉴定.结果 从抽提质粒pET28α-Sj32中PCR扩增出长度约为1 270 bp的Sj32基因片段;重组质粒pGEX-Sj32经双酶切鉴定,获得长度为4 947 bp的载体片段和长度为1 270 bp的Sj32基因片段;以抽提的疫苗质粒pGEX-Sj32为模板进行PCR扩增,得到了长度约为1 270 bp的Sj32基因片段,与预期结果相符.结论 成功构建了日本血吸虫重组Bb(pGEX-Sj32)疫苗.     

Novel molecular mechanism for generating NK‐cell fitness and memory

The mammalian immune system has been traditionally subdivided into two compartments known as the innate and the adaptive. T cells and B cells, which rearrange their antigen‐receptor genes using the RAG recombinase, comprise the adaptive arm of immunity. Meanwhile, every other white blood cell has been grouped together under the broad umbrella of innate immunity, including NK cells. NK cells are considered innate lymphocytes because of their rapid responses to stressed cells and their ability to develop without receptor gene rearrangement (i.e. in RAG‐deficient mice). However, new findings implicate a critical function for RAG proteins during NK‐cell ontogeny, and suggest a novel mechanism by which controlled DNA breaks during NK‐cell development dictate the fitness, function, and longevity of these cells. This review highlights recent work describing how DNA break events can impact cellular differentiation and fitness in a variety of cell types and settings.     

肝内表达的外源性肝细胞生长因子抑制肝细胞凋亡的机制

目的 通过体内基因转染方法在肝细胞中建立持续、高效表达外源性肝细胞生长因子(HGF)的体系,探讨其在肝细胞凋亡中的作用.方法 通过尾部静脉快速注射大量pCMV-HGF质粒,ELISA检测外周血和肝组织中HGF表达的高峰和持续时间.实验动物分为模型组、pcDNA3空质粒保护组、pCMV-HGF质粒保护组和0.9%氯化钠溶液对照组,每组10只,Western印迹检测Caspase-3,tBid、Bax、细胞色素C的表达.所有数据组间均数比较采用单因素方差分析,均数间的两两比较用q检验.结果 在小鼠尾静脉快速大量注射pCMV-HGF 4 h后就有外源性HGF表达,外周血和肝组织中分别于12 h和8 h达高峰,于注射后第6天仍可检测到HGF表达.D-氨基半乳糖胺/脂多糖可诱导明显的细胞凋亡,并导致tBid、Bax、Caspase-3及细胞色素C的表达明显增加.与模型组和pcDNA3空质粒保护组相比,pCMV-HGF保护组tBid、Bax、Caspase-3及细胞色素C的表达明显减少.结论 外源性HGF能抑制小鼠肝细胞凋亡,通过抑制Bid的活性片段tBid的表达,减少下游凋亡蛋白的激活.     

肝细胞生长因子与肝再生增强因子重组表达质粒对大鼠肝纤维化的影响

目的 研究肝细胞生长因子(HGF)与肝再生增强因子(ALR)重组表达质粒对大鼠实验性肝纤维化的治疗作用.方法 建立二甲基亚硝胺肝纤维化模型后的90只S-D大鼠分为空白组、pcDNA3.1治疗组、pcDNA3.1-HGF治疗组、pcDNA3.1-ALR治疗组、pcDNA3.1-HGF与pcDNA3.1-ALR共治疗组、pcDNA3.1-HGF-ALR治疗组,每组15只.各治疗组大鼠每24小时经尾静脉注射1次相应质粒0.1 μmol,连续3次,空白组不接受任何治疗,另取同批次S-D大鼠10只作为对照组.治疗结束后4 d处死大鼠,留取肝组织采用HE染色观察肝脏的病理形态,采用免疫组织化学染色检测增殖细胞核抗原(PCNA)和c-jun的表达.计量资料采用单因素方差分析,两两比较采用SNK检验,计数资料采用Fisher确切概率法分析.结果 空白组和pcDNA3.1治疗组大鼠肝组织有明显的条索状纤维间隔形成,可见假小叶,两组肝纤维化程度差异无统计学意义(x2=0.317,P=1. 000);其他治疗组肝纤维化均有不同程度改善,以pcDNA3.1-HGF-ALR治疗组改善最明显.对照组肝组织PCNA和c-jun表达均较低,其吸光度值分别为8.6±1.9和3.2±1.2;空白组与pcDNA3.1治疗组肝组织PCNA和c-jun表达均增加,其吸光度值分别为24.1±3.0.24.5±4.3与23.8±3.1、24.9±4.2,与对照组比较,差异有统计学意义(均P＜0.01);其他治疗组的PCNA表达显著增加,c-jun表达显著降低,均以pcDNA3.1-HGF-ALR治疗组的变化最明显.结论 重组表达质粒pcDNA3.1-HGF-ALR能较好地改善大鼠实验性肝纤维化,并可能通过促进肝细胞增殖和抑制原癌基因c-jun的表达来发挥其抗肝纤维化作用.     

高温促进含第1类整合子宿主菌的重组效率

目的探讨高温对大肠杆菌BL21(DE3)宿主中第1类整合子重组效率的影响。方法用pUC19为载体构建高表达整合酶的重组质粒pUCINT,随后在pACYC184载体的不同位置分别插入整合子以及氨基糖苷腺苷转移酶(aadA2)基因盒,该重组质粒命名为pACINAD,将这2种相容的重组质粒转化大肠杆菌BL21(DE3),将20株此菌株随机分成2组,每组各10株,分别在41℃和37℃培养24 h后用荧光定量聚合酶链反应(PCR)测定发生重组反应的整合子的拷贝数和总的整合子的拷贝数,前者与后者的比值即为重组效率。结果 41℃大肠杆菌BL21(DE3)宿主中所测得的重组效率为(1.8±0.5)×10-3,在37℃的重组效率为(1.8±0.5)×10-4。41℃组明显高于37℃组,差异具有统计学意义(t=9.266,P<0.01)。结论高温可显著提高由整合酶介导的aadA2基因盒的重组效率。     

Genotoxic evaluation of sodium fluoride in the Somatic Mutation and Recombination Test (SMART)

In this study, genotoxic effect of sodium fluoride (NaF) was investigated in Drosophila melanogaster Somatic Mutation and Recombination Test. Third-instar larvae trans-heterozygous for two genetic markers mwh and flr, were treated at different concentrations (2.5 μg/ml, 5 μg/ml and 10 μg/ml) of the test compounds. After the treatment the observed mutations were classified according to size and type of mutation per wing. For the evaluation of genotoxic effects, the frequencies of spots per wing in the treated series were compared to the control group, which is distilled water. NaF has genotoxic and toxic effects for concentrations of 5 and 10 μg/ml. The present study shows that NaF may have genotoxic and toxic effects.