庆大霉素生物合成基因簇中抗性基因gmrB及gmrA的功能研究

目的 研究绛红色小单孢菌(Micromonospora purpurea)G1008中抗性基因gmrB与gmrA功能,为阐明庆大霉素生物合成机制奠定基础。方法 以红霉素抗性基因ermE分别置换gmrB和gmrA基因,构建突变株GerB102和GerA102。借助TLC和MS分析突变株代谢产物是否变化,同时检测突变株耐受自身代谢产物的能力是否发生变化。结果 突变株GerB102代谢产物没有发生明显变化,仍主要积累庆大霉素C化合物,而GerA102代谢产物发生变化。庆大霉素耐受性试验结果显示,GerB102和G1008耐庆大霉素浓度达到6000U/mL以上,而GerA102不足50U/mL。结论 gmrB既非庆大霉素生物合成的关键基因,也非关键的抗性基因,而gmrA是庆大霉素抗性的关键基因。     

海洋链霉菌OUCMDZ-3434 wailupemycins生物合成基因簇中调控基因功能研究

目的 对海洋链霉菌Streptomyces sp. OUCMDZ-3434中聚酮类化合物wailupemycins生物合成基因簇wal中调控基因walC与walV进行功能探究。方法 通过生物信息学手段对walC和walV功能进行分析,采用PCR-targeting策略分别阻断walC和walV基因,高效液相色谱法（HPLC）分析野生株与突变株在相同条件下发酵产物的差异。结果 生物信息学分析表明walC和walV分别编码TetR家族和MerR家族转录调控因子,成功获得阻断突变株Streptomyces sp. OUCMDZ-3434ΔwalC和Streptomyces sp. OUCMDZ-3434ΔwalV, HPLC分析发现两株阻断突变株中wailupemycin G产量未发生明显变化。结论 walC和walV对wailupemycin类化合物的产生可能并未起到明显调控作用,该类化合物的生物调控机理有待进一步研究。     

通过阻断GntR家族调控基因ygrA挖掘海洋链霉菌Streptomyces sp. OUC6819中抗MDR菌活性次级代谢产物

激活南海红树林来源链霉菌Streptomyces sp.OUC6819菌株中不表达或表达量低的隐性生物合成基因簇,挖掘具有优良多重耐药菌（MDR）抗菌活性的次级代谢产物。方法 通过生物信息学分析推测Streptomyces sp.OUC6819基因组中可能的GntR家族调控子,采用PCR-targeting策略敲除其中的ygrA基因,HPLC分析突变株和野生株的发酵产物的差异,并比较粗提物对5株MDR菌抑制活性。结果 HPLC分析结果表明与野生株相比,突变株中化合物1和化合物2产量分别产量提高了9倍和7倍;突变株发酵液粗提物对其中3株MDR菌抑制活性较野生株明显提高。结论 通过阻断GntR家族调控子ygrA激活了Streptomyces sp.OUC6819菌株中具有抗MDR菌活性次级代谢产物合成基因簇的表达,为从中发掘新的抗MDR菌抗生素奠定了必要基础;同时,将为其他海洋链霉菌中隐性基因簇的激活提供重要参考。     

深海链霉菌Streptomyces somaliensis SCSIO ZH66 ptm生物合成基因簇中orf7功能的探究

目的 探究深海链霉菌Streptomyces somaliensis SCSIO ZH66中编码多环特特拉姆酸大环内酰胺(polycyclic tetramate macrolactams,PTMs)类化合物somamycin A-E的生物合成基因簇ptm中orf7对该基因簇编码产物的影响。方法 采用PCR-targeting策略将ptm基因簇中的orf7进行阻断,然后将野生株与突变株在相同条件下发酵后通过HPLC分析比较代谢产物的差异。 结果 生物信息学分析结果表明orf7的编码产物为细胞色素P450,实验成功得到了阻断突变株Δorf7, 通过发酵产物HPLC分析发现,orf7被阻断后,somamycin D的产量提高,而其14位羰基氧化物somamycin C则不再产生。 结论 说明ptm基因簇中细胞色素P450基因orf7在PTMs生物合成过程中参与了14位碳上的氧化修饰。     

龟裂链霉菌工业菌中zwf1基因的阻断对土霉素生物合成影响

目的运用代谢工程手段对龟裂链霉菌工业菌(Industrial Streptomyces rimosus,SRI)进行基因改造,提高土霉素(oxytetracycline,OTC)产量。方法利用pKC1139质粒阻断SRI基因组中葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase)编码基因zwf1。结果筛选得到一株OTC高产突变株,将突变株与原始菌株进行发酵,发现OTC产量比原始菌株提高了36.2%。结论 SRI基因组中zwf1基因的缺失使细胞合成土霉素的能力增强;龟裂链霉菌中初级代谢关键基因调控会影响次级代谢。     

东方拟无枝酸菌中vcm14基因的敲除对万古霉素合成的影响

PCR-targeting是一种用于敲除或阻断放线菌染色体上某一基因的快速高效的方法 .本研究利用该方法 在大肠埃希菌中构建破坏粘粒cLYLH17(△vcm14::apr),通过结合转移首次敲除东方拟无枝酸菌HCCB10007中万古霉素生物合成基因簇中推测的haloperoxidase基因--vcm14,得到一株双交换阻断突变株A.orientalis dvcm14(Avcm14::apr).该菌不产生万古霉素及其类似物,证实vcm14基因的编码蛋白不是haloperoxidase,且该蛋白不参与万古霉素的卤化反应,可能是万古霉素生物合成途径中的一个关键酶.本研究为阐明万古霉素生物合成途径中的卤化过程提供了有益线索.     

抗生素产生菌核糖体相关基因改变与次级代谢产物的生物合成

利用抗生素抗性筛选方法及定点突变方法可以得到引起抗生素产生菌核糖体相关基因改变的突变株,这些突变对次级代谢产物的生物合成产生深刻影响,如能够显著地提高抗生素的产量也能够产生一些新的次级代谢产物。这种方法不仅对抗生素产生菌的菌种选育具有重要的意义,且为获得新的抗生素提供了新的途径。本文就抗生素产生菌核糖体蛋白基因、核糖体RNA基因、核糖体修饰相关基因及核糖体相关蛋白基因的改变引起次级代谢改变研究进展做简要综述。     

匹马菌素的生物合成研究进展

匹马菌素是纳塔链霉菌产生的一种安全、高效的多烯大环类真菌抑制剂,广泛应用于医药领域与食品工业.作为26元环的糖基化多烯大环聚酮,其生物合成基因簇长度约110kb,包含19个基因,编码5个聚酮合酶、10个修饰和转运蛋白及4个调控因子.本文分析了匹马菌素生物合成的基因基础、多烯聚酮合成过程、氧化和糖基化修饰与调控机理等最新研究进展,展望了利用组合生物合成进行基因簇改造的方案与应用前景.     

海洋芽孢杆菌Bacillus methylotrophicus B-9987中difficidins生物合成基因簇的分析鉴定

目的 对海洋芽孢杆菌B-9987中difficidins生物合成基因簇及其关键基因进行分析、鉴定及功能研究。 方法 通过生物信息学手段对difficidins基因簇的基因组成和功能结构域进行了分析；结合其结构特征及聚酮化合物的生物合成原理,初步推导其生物合成途径；构建了烯酰辅酶A水合酶基因difO双交换阻断突变株,对其功能进行初步验证并对基因簇进行确认。 结果 从B-9987发酵液中分离纯化得到化合物oxydifficidin,鉴定了B-9987中该化合物的生物合成基因簇,其是由位于同一个操纵子的15个基因difA-O所编码的反式酰基转移酶聚酮合酶体系组装而成；difO基因阻断后,oxydifficidin不再产生。 结论 difficidins生物合成基因簇的鉴定为后续研究其生物合成途径及相关基因的功能奠定了基础。     

Difficidins生物合成基因簇中蛋白激酶基因difB功能研究

目的 对海洋芽孢杆菌Bacillus methylotrophicus B-9987 difficidins化合物生物合成基因簇中difB基因功能进行探究。方法 通过生物信息学手段对difB的功能进行预测;构建difB双交换阻断突变株及高表达株;通过高效液相色谱法（HPLC）分析野生株和突变株发酵产物的差异;在大肠杆菌中表达DifB蛋白,以脱磷酸difficidin（3）作为底物,探究DifB的体外功能。结果 得到了ΔdifB双交换阻断突变株及difB高表达株;HPLC分析结果表明,相比较野生株,ΔdifB突变株中difficidins化合物峰不再产生,difB高表达株中difficidins的产量与野生株无显著差别;DifB蛋白不能催化脱磷酸化difficidin（3）发生磷酸化反应。结论 difB是difficidins生物合成所必须的,可能与磷酸基团的组装相关,但磷酸化机制有待进一步研究。     

手指表皮嵴形成的基因作用机制

作者认为胎儿手指皮肤表皮和表皮初级嵴形成受两种不同基因控制,且控制表皮和表皮初级嵴发育的两种基因,由于突变可以相互转化。作者给出描述基因控制过程的微分方程。根据微分方程,阐述了皮纹模式形成的基因作用机制。     

On the Transport of Lipoplexes Through Cystic Fibrosis Sputum

Purpose. The aim of this study was to examine the extent to which plasmid DNA (pDNA) complexed to cationic liposomes diffuse through cystic fibrosis (CF) sputum. The influence of the physical and chemical properties of the sputa was evaluated. We further investigated whether degradation of the sputa by recombinant human DNase I (rhDNase I) enhances the transport. Methods. The transport of lipoplexes was studied through layers of CF sputa placed between the donor and acceptor compartment of vertical diffusion chambers. The content of the acceptor compartment was analyzed by confocal fluorescence microscopy, gel electrophoresis and Southern blotting. The influence of linear DNA present in the CF sputa on the size, surface charge and gene expression of the lipoplexes was evaluated by dynamic light scattering, particle electrophoresis and transfection experiments. Results. Lipoplexes were observed in the acceptor compartments. However, the percent of diffused lipoplexes was low: 0.05% ± 0.01%. It was found that both steric obstruction by the sputa as well as the long distance the lipoplexes have to travel were responsible for this low transport. Surprisingly, the transport occurred better through more viscoelastic sputa. The DNA in the CF sputa also retarded the transport, which was attributed to aggregation of the lipoplexes by the DNA. Finally, rhDNase I moderately enhanced the diffusion of lipoplexes. Conclusions. CF sputum drastically retards the diffusion of lipoplexes. DNA in the sputa aggregates the lipoplexes. This may lower the transport of lipoplexes through the sputa and gene expression. Pretreatment of CF patients with rhDNase I may enhance the efficiency of CF gene therapy, as it allows a better transport of the lipoplexes through the sputum and as it partly removes the sputum which will result in a thinner sputum layer on top of the epithelial cells.     

胎兔与成兔皮肤瘢痕基因差异表达的分析

目的 探讨胎兔及成兔皮肤瘢痕基因表达变化的特征及其可能的生物学意义.方法 建立动物模型,收集胎兔及成兔皮肤愈合处的皮肤组织,用基因芯片技术检测胎兔及成兔皮肤瘢痕的基因表达变化规律.结果 基因芯片高通量地揭示了胎兔及成兔皮肤瘢痕的基因表达变化的信息,差异表达基因有117个,其中下调的基因有28个,上调的基因有89个,涉及十大类基因.结论 胎兔及成兔皮肤瘢痕的基因图谱存在差别,这些基因可能参与了瘢痕的形成过程.     

体内基因突变试验研究进展

遗传毒性研究是新药非临床安全性评价的重要内容之一。由于遗传毒性体外细胞试验不能完全反映整体动物系统对受试物的吸收、分布、代谢和排泄等情况,而且试验结果的假阳性率过高,因此采用体外细胞试验进行药物安全性评价尚存在一定局限性。为了进一步完善对致突变风险的控制,尚需建立更加灵敏的体内基因突变试验方法。本文主要就体内基因突变试验的基本原理、优缺点及基本应用等研究进展进行综述,并对这些体内试验的发展和完善提出一些展望。     

Recent advances in the development of polyethylenimine-based gene vectors for safe and efficient gene delivery

Introduction: Despite the great therapeutic potential of gene therapy for treating critical diseases, the clinical application is limited by lack of safe and effective gene delivery vectors. Nonviral gene vectors have attracted tremendous attention due to the favorable loading capacity and facile manufacture. Among them, polyethylenimine-based gene vectors (PEIs) hold great promise for highly efficient gene delivery.

Areas covered: In this review, we outline the multiple biological barriers associated with gene delivery process and point out several challenges exist in the clinical usage of PEIs. We then provide an overview of the most impressive progresses made to overcome such challenges in recent years, including modifications of PEIs (i.e. to enhance biocompatibility, specific targeting effect, and buffering capacity) and stimuli-responsive strategies (i.e. endogenous and exogenous stimuli) for safe and efficient gene delivery.

Expert opinion: Rational modification of PEIs with diverse functionalized segments or the development of stimuli-responsive PEIs is an appealing strategy to meet some requirements involved in gene delivery. Nevertheless, further optimization by combining the two strategies is needed for the maximized transfection efficiency and minimized side effects, shedding new light on the development of nonviral gene delivery for clinical application.     

PPP1R3基因表达对直肠癌预后的判定价值的研究

目的探讨PPP1R3mRNA基因突变与直肠癌预后的关系。方法采用RT-PCR技术检测53例直肠癌癌肿组织、第一枚淋巴结和复发/转移灶中PPP1R3mRNA基因的表达。结果有62%（33/53）的直肠癌组织中出现PPP1R3mRNA基因的异常,而正常对照结肠壁组织无PPP1R3mRNA基因的异常。依据术后病理所确定的淋巴结有无转移状态,有淋巴结转移者PPP1R3mRNA基因突变率为66%（23/35）,比无淋巴结转移者异常率高,差异有显著性（P〈0.05）;PPP1R3mRNA基因异常率与直肠癌患者的性别、年龄、癌肿的形态及部位无显著性差异（P〉0.05）,与癌细胞分化程度、TNM分期、癌肿的大小、浸润深度、有无局部及远处转移有显著性差异（P〈0.05）。复发/转移组织中PPP1R3mRNA基因异常率更高。结论直肠癌PPP1R3mRNA基因异常与癌细胞的局部浸润和淋巴结转移的有无等生物学行为有一定相关,术前可用于判断直肠癌的局部浸润状况和淋巴结转移的有无、术后用于预测局部复发和淋巴结转移可能起到一定的判断作用,特别是对Ⅱ期者更有意义。     

The role of the disulfide group in disulfide-based polymeric gene carriers

Background: An essential prerequisite for successful gene therapy is the development of safe and efficient gene delivery carriers. For this purpose, cationic polymers have been widely studied as non-viral carriers, but they generally suffer from low transfection efficiency and/or high cytotoxicity. To address these problems, disulfide-based cationic polymers have been designed as intelligent gene carriers that are capable of inducing highly efficient gene transfection with low cytotoxicity. Objective: The present review discusses the effects of the disulfide linker on the gene delivery properties of cationic polymers in relation to various gene delivery barriers. Methods: The literature regarding the gene delivery barriers encountered by polymeric gene delivery is reviewed and discussed in relation to the presence of the disulfide moiety in these gene carriers. Conclusions: The presence of disulfide linkages in cationic polymers can in many aspects favorably influence the gene delivery properties, such as increasing DNA binding ability, enabling de-shielding of ‘stealth’ (PEG) groups, fine-tuning of the buffer capacity for enhanced endosomal escape, improving carrier-unpacking and decreasing cytotoxicity. Therefore, disulfide-based cationic polymers are promising candidates for the next generation of non-viral carriers.     

Stabilized Nonviral Formulations for the Delivery of MCP-1 Gene into Cells of the Vasculoendothelial System

PURPOSE: The purpose of this study was to develop a stabilized non-viral gene transfer system for the efficient delivery and expression of monocyte chemoattractant protein 1 (MCP-1) gene in cells of the vasculoendothelial system. METHODS: Plasmid DNA was condensed with polyethylenimine (PEI), conjugates of PEI with polyethylene glycol (PEG), and PEI conjugates with the membrane-active peptide melittin. Surface charge and particle size of the resulting gene transfer particles were analyzed by laser light scattering. Reporter gene studies and toxicity assays were conducted on smooth muscle cells and endothelial cells of human, porcine, or rat origin. RESULTS: Nonviral gene carriers containing PEI and PEG were developed that could be produced in batches of several milligrams and conveniently stored as frozen samples. Incorporation of PEG into the transfection complex significantly reduced cellular toxicity. The cryoconserved gene transfer particles mediated high expression of luciferase, enhanced green fluorescent protein (EGFP), or secreted alkaline phosphatase reporter genes. Highest reporter gene expression was achieved with PEI polyplexes containing PEG and melittin. The gene for MCP-1 was efficiently delivered into target cells and resulted in expression of up to 125 ng/ml secreted bioactive MCP-1 protein per 50,000 cells. CONCLUSIONS: Gene carriers based on PEI and PEG display reduced toxicity, can be stored in frozen form without loss of biological activity, and can efficiently transfect cells of the vasculoendothelial system. Such gene carriers hold a potential for use in arterial gene transfer and local secretion of MCP-1 as trigger of therapeutic arteriogenesis in arterial occlusion diseases.     

Solid lipid nanoparticles for applications in gene therapy: a review of the state of the art

Importance of the field: Gene therapy represents a new paradigm in the prevention and treatment of many inherited and acquired diseases, including genetic disorders, such as cystic fibrosis, haemophilia and many somatic diseases, such as tumours, neurodegenerative diseases and viral infections, such as AIDS.

Areas covered in this review: Among a large array of non-viral transfection agents used for in-vitro applications, cationic SLNs are the topic of this review, being recently proposed as an alternative carrier for DNA delivery, due to many technological advantages such as large-scale production from substances generally recognized as safe, good storage stability and possibility of steam sterilization and lyophilisation.

What the reader will gain: The authors give some information on the knowledge of intracellular trafficking and SLNs-DNA complex chemical-physical properties reported until now in the literature.

Take home message: The future success of cationic SLNs for administration of genetic material will depend on their ability to efficiently cross the physiological barriers, selectively targeting a specific cell type in vivo and expressing therapeutic genes.     

右旋糖苷衍生物纳米粒对人乳腺癌细胞的转染作用研究

目的以右旋糖苷为起始反应物制备对人乳腺癌细胞具有高转染活性的右旋糖苷衍生物纳米粒非病毒基因载体。方法先将右旋糖苷40与高碘酸钠以不同的配比进行反应,生成醛基含量不同的氧化右旋糖苷;氧化右旋糖苷与精胺以不同的配比反应,共生成9种右旋糖苷衍生物纳米粒;以绿色荧光蛋白基因为报告基因、mda-mb-435人乳腺癌细胞为转染细胞,考察9种合成物的基因转染活性,从中选出转染活性最高的生成物。结果高碘酸钠与右旋糖苷40按摩尔比为0.8∶1反应生成氧化右旋糖苷;该氧化右旋糖苷再与精胺按1∶1的摩尔比继续反应,生成的终产物的转染效率最高。结论制备出了对mda-mb-435人乳腺癌细胞具有高转染活性的右旋糖苷衍生物纳米粒非病毒基因载体。