三链DNA稳定性的影响因素

介绍了碱基组成、碱基修饰或替代、DNA骨架的修饰、DNA配体的结合及反应体系中的盐离子和pH值等因素对三链DNA稳定性的影响。对三链DNA稳定性研究中应注意的几个问题也作了讨论。     

High level expression of human endostatin in Pichia pastoris using a synthetic gene construct

Endostatin, a 20-kDa C-terminal fragment derived from type XVIII collagen, is a potent angiogenesis inhibitor and an antitumor factor. To improve the production of recombinant human endostatin on increasing demand in clinical practice, we constructed an artificial gene encoding its mature peptide sequence in human collagen XVIII. The synthetic gene consisted of 20 codons in preference in methylotropic yeast—Pichia pastoris and was cloned into expression vector pPICZαA; and the recombinant protein was expressed in P. pastoris strain SMD1168 and purified to near homogeneity using heparin affinity chromatography. The amount of expressed recombinant protein in cultural media using described strategy was 80 mg/l in shake flask cultivation and 435 mg/l in high-density bioreactor fermentation. Methylthiazolium assay demonstrated that human endostatin expressed in P. pastoris using artificial synthetic gene of preference in P. pastoris was able to inhibit the acidic fibroblast growth factor-induced proliferation of endothelial cells in vitro.     

青海一株蚕豆根瘤菌的鉴定及抗旱性评价

【目的】研究青海干旱地区蚕豆根瘤菌的遗传多样性,获得与蚕豆品种共生匹配且具有耐旱性的根瘤菌株,促进蚕豆耐旱根瘤菌在青海干旱地区生产中的应用。【方法】以分离自青海干旱地区一株菌株QHCD22为材料,利用细菌形态学、生理生化指标鉴定、Biolog细菌鉴定系统、16S rRNA基因序列分析、全基因组分析等进行菌种鉴定和系统发育分析,进一步通过PEG6000模拟干旱胁迫、盆栽回接干旱胁迫处理及旱作田间接种验证试验对该菌株的耐旱性进行综合评价。【结果】QHCD22菌株属快生型根瘤菌属(Rhizobium),Rhizobium indicum种。随着PEG6000模拟干旱胁迫程度的加剧,在−0.6 mPa这一更低渗透势时菌株存活数量增高,浊度由61.48%上升到69.42%,表现出较强的耐旱性。盆栽试验表明,接种根瘤菌处理(NA)的株高、植株鲜干比、根瘤数、根瘤鲜重、叶绿素含量(SPAD)、叶片相对含水量(RWC)、脯氨酸含量(PRO)、超氧化物歧化酶活性(SOD)、根系活力(TCC)均高于不接种根瘤菌处理(NN),并且在正常供水条件下,NA处理的各指标也均高于NN处理。旱作田间验证试验表明接种该菌株显著提高固氮酶活性,青海13号蚕豆根瘤固氮酶活性由不接种的42.07 C₂H₄ nmol/(g·h)显著增加到221.78 C₂H₄ nmol/(g·h),青蚕14号蚕豆由40.60 C₂H₄ nmol/(g·h)显著增加到109.78 C₂H₄ nmol/(g·h),马牙蚕豆由33.41 C₂H₄ nmol/(g·h)显著增加到643.15 C₂H₄ nmol/(g·h)。接种根瘤菌对于增加产量具有促进作用,其中青蚕14号的增产效果显著,增产幅度达32.3%。【结论】QHCD22菌株可能为快生型根瘤菌属的一个种Rhizobium indicum,具有一定的耐旱性,研究表明接种根瘤菌可以提高蚕豆的耐旱性,尤其对干旱敏感型蚕豆品种增产效果显著,具有潜在的应用前景。     

人恶性疟杂合多肽抗原基因化学合成及克隆

本文报道用固相亚磷酰胺法合成人恶性疟杂合多肽抗原基因。基因全长为216bp,分为10个寡聚核苷酸片段分别合成,然后经T4 DNA连接酶按设计顺序连接成完整的杂合抗原基因,重组到噬茵体M13 mp 18 RF DNA内,转染大肠杆菌JM109。用分子杂交和酶切分析筛选出重组克隆体。经序列分析,证明所合成的人恶性疟杂合多肽抗原基因与所设计完全一致。     

恶性疟原虫74肽抗原基因在减毒鼠伤寒沙门氏菌的表达和家兔免疫应答

利用减毒鼠伤寒沙门氏菌来表达外源抗原并制备口服活菌苗,是疫苗研究的一个新的突破。作者已在减毒鼠伤寒沙门氏菌SL3261以β—半乳糖苷酶(GZ)融合蛋白的形式表达了人工合成的恶性疟原虫74肽基因HGFC。现报道用活菌SL3261(pWRC)(C组)、SL3261(pWR450-1)(R组)分别免疫新西兰家兔,研究其在家兔体内免疫应答的结果。     

恶性疟合成多肽抗原基因在大肠杆菌中表达产物的分离纯化

我们用化学方法合成的两个恶性疟原虫杂合抗原基因,即A基因和B基因〔1.2〕,分别与表达载体pwR450·1重组并转化到大肠杆菌使其表达〔3.4〕。经筛选获得了pwR／A·7和pwR／B·164两个表达较高的克隆菌(经扫描测定表达融合蛋白量分别为菌体总蛋白的8．8％和11．O％)。叉将A基因和B基因串连后与pwR450·l重组并转化到大肠杆菌JMl03株,经筛选鉴定获得了上述两个基因串连的pwR／AB·20克隆菌(表达融合蛋白量为35．8％)。为了进一步研究这3个克隆菌表达蛋白的免疫功能等活性,用8．0mol／L脲处理包涵体,离心沉淀上清液进行DEAE-ephacel柱层析,用Tris梯度缓冲液洗脱,但分离效果不好,而应用PAGE方法进行分离纯化,则可得到电泳纯、稳定又具有免疫原性的产物。     

一种快速构建35S组成型植物双价表达载体的方法

通过对几种常用植物表达载体的35S和Tnos序列进行比对,设计带有酶切位点的引物35S和Tnos,通过PCR克隆了同时带有35S和Tnos序列的目的基因片段,通过定向的酶切、连接获得双价表达载体.此方法简化了植物双价表达载体构建过程,具有明显时间短、效率高、不需要中间载体、检测简单等特点,而且引物对于多数常用35S组成型植物表达载体是通用的,为快速构建35S组成型植物双价表达载体提供了一种新方法.     

Cross-sectional Seroprevalence and Genotype of Hepatitis E Virus in Humans and Swine in a High-density Pig-farming Area in Central China

Hepatitis E virus(HEV) infection is a common public health problem in developing countries. However, the current prevalence of HEV and the relationship of HEV genotype between swine and human within high-density pig-farming areas in central China are still inadequately understood. Here, cross-sectional serological and genotypic surveys of HEV among the 1232 general population, 273 workers occupationally exposed to swine, and 276 pigs in a high-density pig-breeding area, were undertaken by ELISA and nested RT-PCR methods. Anti-HEV IgG was detected in 26.22% of general population and 48.35% of occupational workers. The prevalence of swine serum HEV-Ag was 6.52%. The prevalence of anti-HEV IgG was significantly higher among the workers occupationally exposed to swine than among the general population. An increased HEV seropositivity risk among the general population was associated with either being a peasant or male and was very strongly associated with the increase of age. Among the occupationally exposed group, the prevalence of anti-HEV IgG antibodies increased with age and working years. Among the 30 HEV-IgM-positive people,the infection rates of clerks in the public, peasants, pork retailers, and pig farmers were higher than those of others. A phylogenetic analysis revealed that all the isolates belonged to subgenotype 4 d, and four people and four pigs shared97.04%–100% sequence homology. This study revealed a high HEV seroprevalence among the general population and workers occupationally exposed to swine in the Anlu City, and supports the notion that swine are a source of human HEV infection.     

中国碳排放权交易政策对地区清洁生产意愿的影响

碳排放权交易政策的实施对于企业开展清洁生产,推动地区经济、社会、环境的可持续发展具有重要意义。基于2011—2017年中国省际面板数据,针对国家发改委"两省五市"碳排放权交易试点政策,运用双重差分法实证分析碳排放权交易对中国各地自愿开展清洁生产的影响。研究结果表明:(1)试点地区企业自愿开展清洁生产的比例平均比非试点地区要高出22个百分点;(2)碳排放权交易政策对当前自愿开展清洁生产比例较低的地区影响程度更显著;(3)碳排放权交易试点有利于地区环境的改善,试点地区PM_(10)、S0_2和NO_2的年平均浓度与比非试点地区相比,分别平均下降了0.0288、0.0170和0.0105 mg/m~3。这些结论有助于我国政府科学有序地实施碳排放权交易政策,提升各地企业开展清洁生产的意愿,改善地区空气环境质量。