外源基因表达与代谢超负荷

随着重组DNA技术的出现,各种基因大量地被分离定型,然后在外源宿主细胞内表达。生物技术研究成果的产业化正迅猛发展,如克隆多拷贝的基因或采用高质粒拷贝数的克隆载体;采用在基因序列上游含有翻译信号的载体;选用带有强转录启动子的克隆载体等等。但是,宿主细胞的表达水平是有限的,因为外源基因在宿主细胞内的表达水平是有限的,因为外源基因在宿生细胞内的表达要利用宿主细胞的大量资源,并增加其代谢负荷。这种强加的代谢超负荷,会导致宿主细胞的生物化学和生理学改变,如果这些改变较大,可降低目的蛋白的产生和回收量。该综…     

肠道菌群紊乱对Treg细胞影响的研究进展

人和动物肠道内存在大量的细菌,并且肠道细菌在宿主多功能代谢和免疫系统平衡中至关重要,它们与宿主免疫和代谢系统共同进化。在正常情况下,肠道菌群与宿主处于长期平衡状态,然而当肠道菌群紊乱时,这种平衡将会被打破,并导致免疫应答的改变和各种疾病的发生。Treg细胞是一种表达转录因子Foxp3的调节型T细胞,是促炎和抑炎反应及免疫平衡中重要的组成成分。近年来,大量的研究揭示了Treg细胞、宿主和肠道菌群之间错综复杂的关系,但并没有对动物体内各个部位Treg细胞的表达变化规律做出系统总结。本综述通过总结近年来人们对肠道菌群的研究,综合阐述肠道菌群紊乱时,Treg细胞在肠、外周血、脾脏和肾中的表达变化规律。     

肠道菌群代谢产物在鼠伤寒沙门菌感染中的作用研究进展

人和动物肠道内生存着多种多样的微生物群体,它们与宿主共同进化,对宿主的健康至关重要。肠道菌群可以发酵宿主难以消化的复杂碳水化合物,为宿主肠道细胞提供能量,同时其代谢产物对肠道病原菌沙门菌的感染产生着重要影响。正常情况下,肠道菌群代谢产物如丁酸与丙酸可以抑制沙门菌在肠道中的定植或者毒力基因的表达,而在肠道菌群受到扰乱时,其代谢的琥珀酸盐和1,2-丙二醇等物质却能促进沙门菌增殖。近年来,越来越多的研究揭示了肠道菌群代谢产物对沙门菌感染的影响。本综述通过总结近年来关于鼠伤寒沙门菌入侵时肠道菌群代谢产物改变的研究,综合阐述了肠道菌群代谢产物影响沙门菌感染的机制。     

禽流感病毒NS1蛋白对细胞的影响

NS1蛋白为流感病毒非结构蛋白,只在病毒侵入宿主细胞后产生.目前NS1蛋白对细胞整体水平上的作用仍不清楚,为了解NS1蛋白在病毒感染细胞中的作用,构建了重组质粒pCMV-myc-NS1并将其转染A549细胞,利用双向电泳技术检测了受NS1蛋白调控的宿主蛋白,以期从蛋白质组水平上研究禽流感病毒与宿主细胞间的相互作用.同时,还检测了转染NS1对细胞增殖和细胞周期的影响.结果显示,NS1在细胞中的表达,能够明显引起宿主细胞代谢的变化,并通过阻滞细胞周期的正常进行而减缓细胞的增殖.     

病毒感染影响宿主细胞代谢研究进展

作为专性的细胞内寄生物,病毒没有独立代谢的能力,因此完全依赖于宿主细胞的代谢机制。病毒利用宿主细胞代谢网络提供的能量和生物合成前体物质来驱动其复制、装配和释放。因此,病毒挟持宿主细胞代谢以实现自身的复制和增殖。此外,病毒还可以通过编码辅助代谢基因(auxiliary metabolic genes,AMGs)调控宿主的细胞代谢,影响碳、氮、磷、硫循环,参与微生物驱动的生物地球化学循环。本文主要从细胞葡萄糖代谢、谷氨酰胺代谢、脂肪酸代谢、病毒AMGs调控宿主代谢影响生物地球化学循环4个方面总结病毒感染对宿主核心代谢途径影响的研究,以期为深入理解病毒-宿主相互作用提供参考,也将为通过代谢干预治疗病毒性疾病提供一定的理论依据。     

合成生物学中动态代谢途径调控策略的研究进展

随着合成生物学的发展,越来越多的天然高附加值化学品和大宗化学品可以通过在微生物底盘细胞中构建人工合成途径来进行生产。然而外源途径基因的不平衡表达以及有毒中间代谢产物的积累往往会影响宿主细胞的正常生长,这也是合成生物学研究中的一个共性难题。因此设计可控的基因表达系统,在特定时刻启动或者关闭人工合成途径,对于避免细胞生长和产物合成之间的相互干扰至关重要。重点介绍了最新的动态代谢调控策略及其在代谢途径调控中的应用,并对其发展趋势进行了展望。     

重组微生物生理学

重组微生物生理学主要研究外源基因与宿主的相互作用以及细胞生理状态对这种相互作用的影响。宿主与外源基因的相互作用可发生在复制、分配、表达、代谢等各种水平,主要的分子机制为核酸-核酸、核酸-蛋白质的相互作用,一些小分子化合物可作为信号或效应子参与大分子的相互作用。重组微生物生理学研究主要包括下列三方面工作:1.噬菌体、质粒与宿主的相互作用以及基因工程菌中目的基因的复制、分配和表达规律。2.微生物响答系统的响答及适应的分子机制及其对外源基因调控的影响。3.细胞生长速率对宿主基因和外源基因调控的影响。这些工作融合了分子水平和细胞水平的研究,模糊了分子生物学和生理学的界限,成为基因工程的理论基础之一。美国微生物学会自1987年起把重组微生物生理学列为年会的一个独立专题。 外源基因与宿主的相互作用 噬菌体和质粒等染色体外基因与宿主基因处于一种依赖、互补和竞争的复杂关系。     

疱疹病毒编码的miRNA与病毒潜伏

MicroRNA(miRNA)是一类长度约为22核苷酸的非编码小RNA,能够在转录后水平上对基因的表达进行调节,进而影响细胞活动。病毒依赖宿主细胞进行复制。宿主可以通过免疫系统抵御病毒的入侵,病毒会发展不同的策略用于逃避宿主的免疫。目前,一些病毒已经被证明可以编码miRNA来重塑细胞环境。疱疹病毒是一类双链DNA病毒,该类病毒能够在宿主体内保持长久的潜伏状态。疱疹病毒家族的很多成员都能够表达miRNA。越来越多实验结果表明,疱疹病毒的miRNA对于调节病毒潜伏以及抑制宿主的抗病毒免疫反应方面发挥着重要的作用。该文讨论了疱疹病毒所编码的miRNA的产生过程和功能,希望有助于了解疱疹病毒的潜伏感染以及病毒与宿主的相互作用。     

代谢在宿主抗病毒天然免疫的重要作用

抗病毒天然免疫是宿主抗病毒感染的第一道防线。研究证明,病毒感染时,几乎所有细胞都能诱导I型干扰素(IFN-I)及其下游的干扰素刺激基因(ISG)的表达,进而介导宿主的抗病毒天然免疫。最新研究发现,细胞组分,尤其是脂质代谢几乎可以促进病毒复制周期的所有阶段,包括病毒与宿主细胞的初始相互作用、囊膜与细胞膜融合、病毒组装和出芽等。这些阶段均是宿主抗病毒天然免疫的作用靶点,也是预防和治疗病毒感染的有效途径。因此,细胞代谢(尤其是脂代谢)在病毒感染诱导的天然免疫中必定发挥极为关键的作用。代谢在抗病毒天然免疫中作用的研究,必将为预防和治疗病毒感染新途径和新方法的开发提供新的思路。综述将重点探讨代谢在线粒体、过氧化物酶体和少数ISG参与的抗病毒天然免疫中的作用。     

大肠杆菌外源蛋白表达载体稳定性的研究进展

构建高产、稳定、可靠的质粒载体成为基因重组表达技术的研究重点之一。宿主细胞代谢反应和质粒不稳定性相关信息的缺乏,仍然阻碍着质粒载体的优化,成为限制外源蛋白在大肠杆菌中高效表达的瓶颈之一。主要论述了大肠杆菌外源蛋白表达载体的稳定性,分别从质粒和外源基因的本身特性、重组质粒转化对宿主细胞造成的影响及其他因素等方面阐述了对质粒载体稳定性的影响,同时介绍了相关的解决办法,从而为大肠杆菌表达系统高效表达外源蛋白提供参考。     

Metabolic load and heterologous gene expression

The expression of a foreign protein(s) in a recombinant host cell or organism often utilizes a significant amount of the host cell's resources, removing those resources away from host cell metabolism and placing a metabolic load (metabolic drain, metabolic burden) on the host. As a consequence of the imposed metabolic load, the biochemistry and physiology of the host may be dramatically altered. The numerous physiological changes that may occur often lowers the amount of the target foreign protein that is produced and eventually recovered from the recombinant organism. In this review the physiological changes to host cells, the causes of the phenomenon of metabolic load, and several strategies to avoid some of the problems associated with metabolic load are elaborated and discussed.     

The effects of G418 on the growth and metabolism of recombinant mammalian cell lines

It is widely reported that the growth of recombinant bacteria and yeast is adversely affected by increased metabolic load caused by the maintenance of plasmid copy number and recombinant protein expression. Reports suggest that recombinant mammalian systems are similarly affected by increased metabolic load. However, in comparison to bacterial systems relatively little information exists. It was the aim of this study to test the effects of recombinant gene expression on the growth and metabolism of two industrially important cell lines. A BHK and CHO cell line were stably transfected with the human gastric inhibitory peptide (h-GIP)and glucagon receptor respectively. Selection was by way of the neomycin resistance (neo ^r) gene using G418.The growth and metabolism of both cell lines was affected by the presence of G418 in a manner indicative of increased metabolic load and which appeared to be caused by over-expression of the neomycin resistance protein. The two cell lines differed in their metabolic response to G418, which suggested that some cell lines or clones may be better able to tolerate a metabolic load than others. Growth under increased metabolic load was affected by medium composition with serum, insulin and glutamine concentration as influencing factors. Implications for the use of G418 are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Use of culture fluorescence as a sensor for on-line discrimination of host and overproducing recombinant Escherichia coli

The culture fluorescence of two recombinant Escherichia coli strains with high plasmid copy number were studied and compared to both the host and low copy number varieties of the corresponding strains. Culture fluorescence data are related to the concentration of reduced intracellular nicotinamide adenine dinucleotide within a cell, and can therefore be used as a means for detecting changes in metabolic states. Correlation curves relating culture fluorescence to biomass show that the recombinant system maintains a larger pool of intracellular NADH at high plasmid copy numbers than either the host or the recombinant system at low copy numbers. These results demonstrate the ability of a fluorescence probe to detect differences in the metabolic demands made on an over-producing recombinant organism.     

重组大肠杆菌高密度发酵研究进展

重组大肠杆菌的高密度发酵是提高基因工程产品产量的一个非常有效的手段,是现代发酵工程研究的一个热点。本文就高密度发酵中影响重组大肠杆菌发酵产率的几个因素,包括宿主菌、培养基、培养条件、补料方法以及高密度发酵过程中存在的问题和对策加以讨论,着重探讨了高密度下大肠杆菌产生的有害代谢副产物———乙酸的产生机制、抑制作用机理,以及控制乙酸积累的技术方法 。     

大豆异黄酮代谢途径在大肠杆菌中的构建及表达

自然界异黄酮合成途径主要存在于豆科植物中。以微生物为宿主研究异黄酮代谢,则需要将整个相关代谢途径的多酶体系组装到工程菌种,从而进行表达及代谢研究,这就需要用到多基因的转化和共表达技术。综合应用了多基因单载体和多基因多载体方法,将大豆异黄酮代谢途径中的五个关键酶基因导入到大肠杆菌中,对异黄酮代谢途径在大肠杆菌中的构建和表达进行了研究和探索,获得了含有五个外源基因的重组大肠杆菌;重组菌经IPTG诱导,以L-酪氨酸为底物进行发酵,发酵产物经过HPLC测定,结果表明和空白对照相比有新的代谢产物生成,初步断定为异黄酮类化合物。     

Rational design of an improved induction scheme for recombinant Escherichia coli

In Escherichia coli, strong overexpression of a recombinant protein has been shown to be deleterious due to a heavy metabolic burden on the host cell, which may completely cease cell growth before maximum product accumulation has occurred. Aiming at a reduction of very high product formation rates, we engineered E. coli strains by mutating the Leloir pathway for galactose metabolization, so that galactose can be utilized to induce lac derived promoters. The induction with galactose was effective in every strain and expression construct tested, and it reduced the metabolic burden on a highly overproducing clone so that cell growth and product accumulation could be maintained for several generations.     

代谢工程与重组大肠杆菌的发酵

利用代谢工程可以在重组大肠杆菌的改良中减少代谢副产物乙酸的累积,优化代谢系统,利于重组蛋白质的高表达以及重组菌的高密度发酵。应用代谢工程改良重组大肠杆菌主要包括阻断乙酸产生的主要途径、限制糖酵解途径上的碳代谢流、将过量的丙酮酸转化为其它低毒的副产物以及对碳代谢流进行分流等几个方面的工作。     

Plasmid-encoded protein: the principal factor in the "metabolic burden" associated with recombinant bacteria

Experimental elucidation of the metabolic load placed on bacteria by the expression of foreign protein is presented. The host/vector system is Escherichia coli RR1/pBR329 (amp(r), cam(r), and let(r)). Plasmid content results, which indicate that the plasmid copy number monotonically increases with decreasing growth rate, are consistent with the literature on ColE1-like plasmids. More significantly, we have experimentally quantified the reduction in growth rate brought about by the expression of chloramphenicol-acetyl-transferase (CAT) and beta-lactamase. Results indicate a nearly linear decrease in growth rate with increasing foreign protein content. Also, the change in growth rate due to foreign protein expression depends on the growth rate of the cells. The observed linear relationship is media independent and, to our knowledge, previously undocumented. Furthermore, the induction of CAT, mediated by the presence of chloramphenicol, is shown to occur only at low growth rates, which further increases the metabolic load.Results are vdelineated with the aid of a structured kinetic model representing the metabolism of recombinant E. coli. In this article, several previous hypotheses and model predictions are justified and validated. This work provides an important step in the development of comprehensive, methabolically-structured, kinetic models capable of prediciting optimal conditions for maximizing product yield.