无活性放线菌野生株抗肿瘤活性突变株的自发突变抗性筛选和化学诱变抗性筛选

目的由无活性放线菌野生株转化获取活性突变株,为药源活性产物研究拓展新菌株资源。方法以海洋来源无活性放线菌野生株L35-1为出发菌株,通过自发突变抗性筛选以及化学诱变结合抗性筛选,筛选获得抗性突变株。采用MTT法检测突变株发酵样品对K562细胞的抑制活性。结果通过自发突变抗性筛选,得到新霉素抗性突变株114株、链霉素抗性突变株68株,其中7株新霉素抗性突变株和3株链霉素抗性突变株样品对K562细胞有抑制作用,在100μg/ml浓度下抑制率大于20%。通过化学诱变结合抗性筛选,得到硫酸二乙酯诱变链霉素抗性突变株41株、硫酸二乙酯诱变新霉素抗性突变株32株、亚硝基胍诱变新霉素抗性突变株46株,其中1株硫酸二乙酯诱变链霉素抗性突变株和1株亚硝基胍诱变新霉素抗性突变株有抗肿瘤活性,100μg/ml样品对K562细胞的抑制率大于20%。结论上述结果初步表明,无活性放线菌野生株可通过抗性筛选转化为活性突变株,因此有可能成为筛选获取药源活性新菌株的重要原始资源。     

海洋来源放线菌无活性野生株的链霉素抗性抗肿瘤活性突变株新产代谢产物研究

目的研究阐明无活性放线菌野生株L35-1来源硫酸二乙酯（DES）诱变链霉素抗性抗肿瘤活性突变株D2s4-1新产代谢产物及其抗肿瘤活性。方法组合利用活性跟踪与微量预试先导-放大实验制备的实验模式,通过与原始菌样品直接对照,在快速确定差异活性斑点基础上,组合利用各种色谱技术,分离纯化突变株新产代谢产物。根据理化性质和波谱数据鉴定化合物结构。采用MTT法测定抗肿瘤活性。结果从突变株D2s4-1发酵物中分离鉴定了6个该突变株新产代谢产物,即1,9-二甲酯吩嗪（1）、2-羟基苯乙酰胺（2）、2-吡咯甲酸（3）、大豆黄素（4）、环（4-羟基-脯氨酸-亮氨酸）二肽（5）和尿嘧啶（6）。其中1、3、5和6有一定抗肿瘤活性,浓度为100μg/ml时对K562细胞的抑制率分别为33.3%、16.3%、33.3%和21.1%。结论阐明了抗肿瘤活性突变株D2s4-1的6个新产物,其中4个为活性产物。化合物1为新天然产物,其抗肿瘤活性亦属首次筛选发现。用化学诱变组合抗性筛选技术从无活性放线菌野生株转化获取的活性突变株可供筛选新产活性产物,从而拓展放线菌药源活性新菌株资源。     

产紫青霉G59的超声诱变活性突变株新产抗肿瘤活性产物

目的阐明无活性真菌野生株产紫青霉G59的超声诱变活性突变株N3001d1-1新产抗肿瘤活性产物。方法采用活性跟踪与高效硅胶薄层检测分析相结合的实验方法,组合利用各种色谱技术,通过将突变株样品与原始菌直接比对分离纯化突变株新产活性产物。根据理化性质和波谱数据鉴定化合物结构。用MTT法测试抗肿瘤活性。结果从突变株N3001d1-1发酵物中分离鉴定了2个突变株新产抗肿瘤活性产物,即橘霉素（citrinin）（1）和fructigenine A（2）。化合物1和2对K562细胞均呈抗肿瘤活性,抑制K562细胞的IC50分别为50.6μg/ml和69.1μg/ml。结论化合物1和2均为原始真菌产紫青霉G59所不生产的突变株N3001d1-1新产抗肿瘤活性次级代谢产物。研究结果表明,超声诱变技术可用于改造真菌无活性野生株的次级代谢功能并从中筛选获取活性突变株,从而拓展真菌药源活性新菌株资源。     

姥鲨共附生微生物抗菌活性筛选及系统发育分析

目的 以深海姥鲨鱼鳃和胃肠道共附生微生物为研究对象,对可培养微生物进行分离、纯化和初步鉴定,筛选抗菌活性,为寻找天然活性产物奠定基础.方法 采用倍比稀释涂布和划线法结合分离纯化可培养微生物;以枯草芽孢杆菌、金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌、产气肠杆菌、白色念珠菌为抗菌活性筛选模型,利用琼脂块法和滤纸片法筛选纯培养菌株抗菌活性;利用形态观察、生理生化反应和16S rRNA技术对抑菌活性进行初步鉴定.结果 从姥鲨鱼鳃和胃肠道分离得到的可培养微生物36株,其中来自鱼鳃5株,来自胃肠道31株;菌株统计表明:真菌7株,占19%;放线菌1株,占3%;细菌28株,占78%.对23株来自姥鲨胃肠道的细菌进行抗菌活性筛选,15种表现抗(抑)菌活性,占分离菌株的65%.他们分别隶属于芽孢杆菌属(Bacillus)5株,葡萄球菌属(Staphylococcus)6株,短波单胞菌属(Brevundimonas)2株,嗜冷杆菌属(Psychrobacter)2株.结论 利用常规微生物学技术可以对姥鲨胃肠道和鱼鳃等部位存在共附生微生物分离和纯化,其中超过50%的菌株表现抗(抑)菌效果.     

隐匿性HBV感染相关S基因突变对HBsAg检测的影响及其机制

目的 探讨隐匿性HBV感染(OBI)相关S基因突变对多种抗-HBs及HBsAg临床检测试剂的反应性.方法 9种代表性S基因突变株(M1-M9,其中M1-M5为本课题组首次报道)分离自1例OBI患者和3例OBI献血人员血清.分别采用野生型及突变型S基因重组质粒转染中国仓鼠卵巢细胞,分析9种突变株HBsAg表达及其突变蛋白对抗体和检测试剂反应的影响.结果 采用HBsAg罗氏诊断试剂Elecsys定量检测9种突变株胞内HBsAg水平,均较野生株的水平明显下降;相同样品用抗-His抗体检测显示仅M1、M6和M7的HBsAg-His融合蛋白表达水平与野生株比较明显降低.6种抗-HBs与各突变HBsAg的反应性结果(S/CO值)显示,与野生株相比,多数突变株与6种抗-HBs的反应性明显降低,M1、M4、M7和M9产生的HBsAg与抗体4及M9产生的HBsAg与抗体6反应性最差(S/CO＜1).6种商品化HBsAg临床试剂检测结果显示,多数突变株与6种HBsAg试剂反应性较野生株明显降低(P＜0.05),ELISA试剂D、E和F漏检率分别为11.1％、22.2％和55.6％.结论 本实验涉及的突变HBsAg与抗-HBs的结合力降低是引起OBI表现的主要原因之一.当前临床常用HBsAg检测试剂对突变HBsAg的检测能力存在明显不足,亟待提高.     

利用我国“90105”科学返回卫星搭载微生物材料的试验观察

利用我国“９０１０５”科学返回卫星，对１０个属、１３个种２４株具有实际应用价值的微生物材料进行了空间飞行条件下生长与代谢性状的研究。结果表明：经过８ｄ的飞行，多数菌株能够存活；一些细菌和放线菌的生长速度加快，生长量增加；芽孢杆菌提前产生出芽孢，伴孢晶体数量增加；经分析多种生化性状，除一株肇庆曲霉菌α－淀粉酶、果胶酶的活性有显著提高外，多数菌株无明显变化；猴头、灵芝等大型真菌仍能产生出正常的食药兼用子实体。     

推理选育林可霉素高产菌株

目的推理选育林可霉素高产菌株。方法根据林可霉素的生物合成途径,对其产生菌林肯链霉菌(Streptmyces Lin-colnsis)进行连续递进推理选育,出发菌株SL369经过UV诱变和多次单菌落分离,筛选林可霉素、L-酪氨酸和α,D-吡喃半乳糖抗性突变株。结果获得高产菌株SL-42,生产能力提高48%,上罐发酵证实,达7 900μ.ml-1以上,为企业赢得较大经济效益。结论推理选育可得林可霉素高产菌株,且高产性能及其遗传特性较为稳定。     

重组人β-防御素3对革兰阴性菌的抗菌活性及其与细菌耐药性的关系

目的 研究重组人β-防御素3(rhBD-3)对临床呼吸道分离的革兰阴性菌(GNB)菌株的体外抗菌活性,分析rhBD-3对GNB的最低抑菌浓度(MIC)与GNB对常用抗菌药物的耐药性间的关系.方法 从呼吸科重症监护病房(RICU)不同时期住院患者采集的下呼吸道痰液中培养、分离、鉴定获得GNB菌株,包括铜绿假单胞菌(20株)、鲍曼不动杆菌(15株)和肺炎克雷伯杆菌(14株).采用琼脂糖弥散抗菌法测定rhBD-3对菌株的MIC.在参与检测的抗菌药物中,计算各株细菌共对几种抗菌药物耐药,并计算这些抗菌药物占所有受检抗菌药物的比例,称为各株细菌的耐药比例.采用Pearson线性相关性检验分析菌株的耐药比例与rhBD-3的MIC间的关系.结果 rhBD-3对所测试的GNB菌株均表现出一定的抗菌活性(MIC 8～64μg/ml),且表现出抗菌活性随浓度增高而增强的趋势.rhBD-3对肺炎克雷伯杆菌、铜绿假单胞菌、鲍曼不动杆菌3种菌株的MIC与菌株对临床常用抗菌药物的耐药性间无明显的相关性(分别P=0.921、r=0.087,P=0.711、r=0.076,P=0.162、r=0.038).结论 rhBD-3对呼吸道分离的3种GNB菌株均有抗菌活性,且这种抗菌活性与菌株对临床常用抗菌药物的耐药特性无明确相关性.     

Avermectin B1a高效突变株的选育及发酵条件的优化

目的筛选获得Avermectins B1a高效突变菌株。方法通过离子注入诱变、紫外线与氯化锂复合诱变法处理阿维链霉菌出发菌株N-5-9。获得高效突变株,再对其进行发酵条件的优化,寻找最适发酵培养条件。结果筛选获得Avermectins高效突变菌株A-30-6,总效价达到6988.2μg·ml-1,B1a含量达到79.4%,较出发菌株N-5-9的B1a组提高46.3%。结论利用离子注入诱变、紫外线与氯化锂复合诱变方法,诱变效果显著,再进行发酵条件的优化,获得目的菌株。     

HBV表型耐药方法的建立及临床分离株的表型耐药分析

目的建立HBV表型耐药分析方法 ,体外培养慢性乙型肝炎患者血清HBV分离株并系统分析其对拉米夫定(LAM)的敏感性。方法从1例LAM耐药慢性乙型肝炎患者血清中提取HBV DNA,PCR扩增RT基因,克隆到pGEM-Teasy载体中,随机挑选10个克隆进行DNA序列测定,并分析RT区LAM耐药相关的突变位点。用XhoI和NcoI双酶切pGEM-Teasy-RT及pTriEx-HBV(C),构建1.1倍HBV野生株和LAM耐药突变株的重组载体pTriEx-wRT和pTriEx-mRT,转染人肝癌细胞系HepG2细胞。转染60h后加入不同浓度(0、0.01、0.1、1、10、100μmol/L)的LAM,药物连续作用5d后,抽提病毒核心颗粒HBV DNA,通过实时荧光PCR及Southern blotting检测不同药物浓度作用下HBV DNA的复制水平。结果成功建立体外HBV表型耐药分析方法。野生未突变株重组载体pTriEx-wRT转染HepG2细胞后,随着LAM浓度的升高,HBV DNA水平明显下降,而突变株重组载体pTriEx-mRT转染HepG2细胞后,HBV DNA水平无明显下降,IC50值与野生株相比增加了2500倍。结论建立的HBV表型耐药分析方法对监测HBV耐药性具有可行性。     

Critical role of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of rejoining following gamma-radiation-induced damage to pMTa4 DNA in E. coli

PURPOSE: This study was undertaken to understand the roles of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of the process following exposure of E. coli to gamma-radiation in vivo. MATERIALS AND METHODS: A plasmid DNA construct, pMTa4, was transformed into isogenic repair proficient (wild) and deficient (recF and recA) E. coli strains and gamma-irradiated up to 30 Gy in vivo. The plasmid DNA was isolated under repair non-permissive (R-)and permissive (R+) conditions and analyzed by gel electrophoresis for the yields of single strand breaks (SSB) and double strand breaks (DSB) and their repair. The clonogenic survival of the E. coli was also recorded. The effects of gamma-irradiation on recA reconstituted with cell free extract of wild strain or ultra-violet (UV)-irradiation were also monitored. RESULTS: None of the strains used in this investigation showed effects of radiation-induced oxidative base damage. The dose dependent increase in SSB and DSB on pMTa4 in wild and recF mutants in R- condition were abolished upon repair incubation. The recA mutant exhibited a disturbed yield of SSB and DSB along with formation of gamma-radiation-induced 'ladder'. The 'ladder' was not observed after repair incubation, UV-irradiation or gamma-irradiation in presence of cell-free extract of wild strain. The survival of recA mutants was seriously compromised. CONCLUSIONS: Wild, recF and recA strains of E. coli could repair gamma-irradiation-induced oxidative damage to base or nucleotide (NT) in vivo. In absence of either RecA or RecF proteins, efficiency of rejoining of strand went down; RecA proteins seemed more critical than RecF in this. High fidelity or correct rejoining of strand breaks, on the other hand, seemed to require simultaneous presence of both RecA and RecF proteins.     

MiR30b与小鼠Bach2相互作用的研究

目的研究B细胞末端分化过程中miR30b新的作用靶点。方法经生物信息学分析,利用特异性引物以及定点突变引物从小鼠c DNA中分别扩增大小为530、361和189bp三个目的片段,胶回收并对361和189bp进行拼接,获得野生型、突变型小鼠Bach2 mRNA特异性片段,分别与pmir GLO载体连接,构建野生型、突变型重组荧光素酶报告基因质粒pmir GLO-m Bach2、pmir GLO-m Bach2 mt;与miR30b共转染至HEK293 T细胞,分析其荧光素酶活性。结果重组荧光素酶报告基因质粒经双酶切及测序证实构建正确;共转染后,与pmir GLO+miR30b组相比,pmir GLO-m Bach2+miR30b组的荧光素酶活性明显降低(P0.05),而pmir GLO-m Bach2 mt+miR30b组的荧光素酶活性则无明显改变(P0.05)。结论小鼠Bach2 mRNA是miR30b的靶点。     

Use of 111In-labelling to follow tissue distribution of Candida albicans in mice.

Two strains of Candida albicans, a wild type and a derived mutant, were labelled with 111Inoxine. Labelled cells were injected into mice and tissue distribution patterns were determined from 0.5 to 48 h. During the first 4-h post-injection phase, remarkable differences in tissue distribution were observed between the two strains. Radiolabelling of C. albicans with 111Inoxine is shown to be a much more reliable method for determining early tissue distribution patterns in infected animal models than culturing the infected tissue.     

人野生型和突变型CITED2真核表达质粒的构建与表达

目的 构建人转录辅助因子CITED2突变型(c.573-578del6)及野生型真核表达质粒并检测两种重组质粒CITED2蛋白的表达情况.方法 分别以健康儿童和CITED2基因突变的先天性心脏病患儿血细胞DNA为模板,PCR定向克隆扩增野生型和突变型CITED2编码链,分别T/A克隆至pMD19-T simple质粒上,筛选出野生型和突变型CITED2的T载体重组质粒.应用DNA重组技术将T载体重组质粒上的CITED2基因片段亚克隆入真核表达载体pEGFP-C1,构建pEGFP-C1-wtCITED2和pEGFP-C1-mtCITED2真核表达质粒,并分别转染至HEK293细胞,24h后在荧光显微镜下观察质粒转染情况,48h后应用流式细胞仪检测转染效率,Western blotting检测CITED2蛋白的表达.结果 成功构建了人野生型pEGFP-C1-wtCITED2和突变型pEGFP-C1-mtCITED2真核表达质粒.转染至HEK293细胞后24h,在荧光显微镜下可观察到各转染组EGFP的表达,转染后48h流式细胞仪检测转染效率为50％ ～ 60％,Western blotting检测可见CITED2蛋白与EGFP的融合表达.结论 成功构建了人转录辅助因子CITED2突变型及野生型真核表达质粒,并检测到CITED2蛋白的表达.     

E4F1真核表达载体的构建及与p53的相互作用

目的：构建E4 F1野生型及缺失32～81位氨基酸的真核表达载体,检测其与p53的相互作用及对下游基因p53的调节。方法分别用普通PCR和重组PCR方法从乳腺文库中扩增E4F1野生型编码序列及缺失32～81位氨基酸的突变型序列,分别将其以正确相位构建到pXJ40-MYC载体中,得到重组质粒MYC-E4F1和MYC-E4F1（Δ32-81）,分别转化大肠杆菌DH5α,重组质粒经酶切鉴定并转染293 T细胞, Western印迹检测蛋白的表达。将MYC-E4F1和MYC-E4F1（Δ32-81）质粒与FLAG-p53质粒共转染293T 细胞,免疫共沉淀实验检测其相互作用,野生型及缺失突变型表达载体共转染骨肉瘤细胞U2OS,检测其对下游基因p53的调节。结果 E4F1重组质粒及其突变型构建成功,在293 T细胞中鉴定表达正确,野生型及突变型E4 F1均与p53存在相互作用,突变型的结合能力增强,野生型E4F1升高p21蛋白表达水平,而突变型不改变p21蛋白水平。结论成功构建E4F1野生型及缺失32～81位氨基酸的突变型真核表达载体,二者都能与p53相互作用且突变型结合能力更强,野生型E4F1升高基因p53的靶基因p21的蛋白表达水平,而突变型不改变靶基因p21的蛋白水平。该研究为进一步研究E4 F1对p53的调节及其机制奠定了基础。     

Improvement of organophosphorus hydrolase activity toward nerve agents by amino acid substitutions

The hydrolytic activities of organophosphorus hydrolase (OPH) toward nerve agents were investigated by using mutant OPH enzymes constructed by site-directed mutagenesis of the gene. The amino acids at position 136, 254, and 257 are known to influence the enzyme activity. The nucleotide sequencing of the OPH gene cloned in our previous study revealed that the deduced amino acid at position 254 was tyrosine (Tyr) instead of histidine (His) that was reported to reside at this position by other research groups. In the present study, such key amino acids were substituted to construct five mutant enzymes, and their hydrolytic activities were compared with that of the wild type enzyme. The activity assays proved that the substitution of Tyr at 254 to His led to the remarkable enhancement of the activities toward paraoxon and also toward VX, which could be decomposed by the wild type OPH only slightly. This mutant enzyme hydrolyzed most of the nerve agents almost completely, and approximately half of the VX during 20 min when the enzyme was activated with 10 mM CoCl₂. The cobalt ion could activate the mutant OPH more efficiently than zinc ion, but the difference of the activation ability was not significant when other mutant OPH enzymes were activated with these divalent metal ions. The present results suggest that a practical decontamination system for nerve agents could be established using the mutant OPH with enhanced activity.     

Effect of 2.45 mT sinusoidal 50 Hz magnetic field on Saccharomyces cerevisiae strains deficient in DNA strand breaks repair

Purpose:?To investigate whether extremely-low frequency magnetic field (MF) exposure produce alterations in the growth, cell cycle, survival and DNA damage of wild type (wt) and mutant yeast strains.

Materials and methods:?wt and high affinity DNA binding factor 1 (hdf1), radiation sensitive 52 (rad52), rad52 hdf1 mutant Saccharomyces cerevisiae strains were exposed to 2.45?mT, sinusoidal 50?Hz MF for 96?h. MF was generated by a pair of Helmholtz coils. During this time the growth was monitored by measuring the optical density at 600?nm and cell cycle evolution were analysed by microscopic morphological analysis. Then, yeast survival was assayed by the drop test and DNA was extracted and electrophoresed.

Results:?A significant increase in the growth was observed for rad52 strain (P?=?0.005, Analysis of Variance [ANOVA]) and close to significance for rad52 hdf1 strain (P?=?0.069, ANOVA). In addition, the surviving fraction values obtained for MF-exposed samples were in all cases less than for the controls, being the P value obtained for the whole set of MF-treated strains close to significance (P?=?0.066, Student's t-test). In contrast, the cell cycle evolution and the DNA pattern obtained for wt and the mutant strains were not altered after exposure to MF.

Conclusions:?The data presented in the current report show that the applied MF (2.45?mT, sinusoidal 50?Hz, 96?h) induces alterations in the growth and survival of S. cerevisiae strains deficient in DNA strand breaks repair. In contrast, the MF treatment does not induce alterations in the cell cycle and does not cause DNA damage.