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大肠杆菌中外源基因表达的研究进展 总被引:10,自引:0,他引:10
工程技术已使许多生物活性蛋白基因在大肠杆菌中中得到表达,但不同外基因表达效率并异很大,本文综述了影响外源基因表达的一些因素,以便采取有效方法提高外源基因在大肠杆菌中的表达效率。 相似文献
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外源基因在大肠杆菌中表达的研究进展 总被引:16,自引:0,他引:16
大肠杆菌是外源基因表达的首选体系.大肠杆菌中外源蛋白可定位于胞内、周质或胞外培养基中.按照重组蛋白的可能命运,综述了最近几年大肠杆菌表达体系的研究进展. 相似文献
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pBV220载体中外源基因高效表达的自动化设计 总被引:1,自引:0,他引:1
pBV220载体是国内科学家构建的原核系统表达载体,目前仍在广泛应用.但是,实现 外源基因的高效表达需要综合考虑诸如RNA二级结构等多种因素,极其耗时费力.为此,基于我们提出的pBV220载体中外源基因高效表达数学模型,编写了外源基因高效表达的自动化设计软件,并可定性用于原核系统其它载体中外源基因表达水平分析,最终为加快实验进程提供帮助 相似文献
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自七十年代基因工程建立以来,对基因的分离、导入及其在受体中的表达等方面进行了大量的研究,也取得了可喜的成果.本文把能表达产物并可检测的外源基因重组质粒导入大肠杆菌不同受体菌株中,观察了不同受体对外源基因表达的影响.发现同一基因在不同的大肠杆菌受体中的表达量有很大差异,这说明不同受体对外源基因表达的影响是不同的。 相似文献
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外源基因的表达效率低是蓝藻基因工程发展的瓶颈之一,T7 RNA聚合酶表达系统实现了大肠杆菌中外源基因的高效表达,蓝藻与大肠杆菌同为革兰氏阴性菌,具有较高的遗传同源性,在蓝藻中构建T7 RNA聚合酶表达系统有可能提高外源基因在蓝藻中的表达效率。为了在鱼腥藻7120中构建T7 RNA聚合酶表达系统,采用重叠延伸PCR技术和酶切连接等方法构建能够表达T7 RNA聚合酶的定点整合载体pEASY-T1-F1-TacT7RNAPCmR-F2以及由T7启动子驱动hG-CSF基因表达的穿梭表达载体pRL-T7-hG-CSF;采用电击转化法将定点整合载体导入野生型鱼腥藻中,通过三亲接合的方法将穿梭表达载体转入已定点整合T7 RNA聚合酶的转基因鱼腥藻中。利用PCR技术鉴定外源基因在蓝藻中的存在;RT-PCR方法检测外源基因在蓝藻中的转录情况;Western blotting实验检测外源基因在蓝藻中的蛋白表达情况。结果表明两种载体构建成功,T7 RNA聚合酶基因和hG-CSF基因被转入鱼腥藻中,两个基因均在藻中表达,T7 RNA聚合酶表达系统在鱼腥藻中构建成功,与传统蓝藻表达系统相比,文中在鱼腥藻中构建的T7表达系统使hG-CSF基因的表达量提高2倍。该表达系统将为蓝藻基因工程的应用提供更优的工具,将促进蓝藻作为底盘细胞在合成生物学等领域的发展。 相似文献
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MAR的结构、功能及其应用研究现状 总被引:2,自引:0,他引:2
真核细胞表达外源基因克服了原核细胞表达外源基因的种种不足(如mRNA的成熟、翻译后的加工),因而得到广泛应用。但真核细胞表达外源基因也有不足之处,即表达效率低。造成真核细胞外源基因表达效率低的原因有很多,如基因结构、表达载体、调控元件等,而宿主细胞染色质DNA对外源基因的影响是一个不可忽视的原因。人们在通过载体将外源基 相似文献
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类弹性蛋白多肽(ELP)为含有人工合成的ELP60基因的表达载体pRELPN,能促使外源基因在大肠杆菌中的高表达。当ELP60在大肠杆菌表达载体pET28a的多克隆位点被克隆后,其自身的表达低,也不与目的基因构成ELP融合蛋白质,而是促进克隆在ELP60基因后的含起始密码ATG的外源目的基因独立高表达。外源目的基因表达量占宿主蛋白的20% ~ 60%,比用pET28a载体表达的外源基因表达量高2~10倍。此类表达载体pRELPN适合于表达包括抗体、抗原、酶、重组蛋白质、多肽及ELP融合蛋白质等的外源基因的独立高表达。这些结果表明,pRELPN代表了一种有效的表达载体,有助于解决在原核表达中,所受限的普通载体对外源基因低表达或不表达所导致的不能产业化的问题。 相似文献
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Arne R Strøm 《Journal of biosciences》1998,23(4):437-445
Glycine betaine is known to be the preferred osmoprotectant in many bacteria, and glycine betaine accumulation has also been
correlated with increased cold tolerance. Trehalose is often a minor osmoprotectant in bacteria and it is a major determinant
for desiccation tolerance in many so-called anhydrobiotic organisms such as baker's yeast(Saccharomyces cerevisiae). Escherichia coli has two pathways for synthesis of these protective molecules; i.e., a two-step conversion of UDP-glucose and glucose-6-phosphate
to trehalose and a two-step oxidation of externally-supplied choline to glycine betaine. The genes governing the choline-to-glycine
betaine pathway have been studied inE. coli and several other bacteria and higher plants. The genes governing UDP-glucose-dependent trehalose synthesis have been studied
inE. coli andS. cerevisiae. Because of their well-documented function in stress protection, glycine betaine and trehalose have been identified as targets
for metabolic engineering of stress tolerance. Examples of this experimental approach include the expression of theE. coli betA andArthrobacter globiformis codA genes for glycine betaine synthesis in plants and distantly related bacteria, and the expression of theE. coli otsA and yeastTPS1 genes for trehalose synthesis in plants. The published data show that glycine betaine synthesis protects transgenic plants
and phototrophic bacteria against stress caused by salt and cold. Trehalose synthesis has been reported to confer increased
drought tolerance in transgenic plants, but it causes negative side effects which is of concern. Thus, the much-used model
organismE. coli has now become a gene resource for metabolic engineering of stress tolerance. 相似文献
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Host cell physiology is known to play a crucial role in the expression of foreign genes in heterologous systems. Expression
of archaeal genes in anaerobic or semi-anaerobic growth conditions of E. coli has been previously reported to be a means of improving solubility of some proteins. Here, we report that some of the Rosetta
strains of E. coli, which harbour the rare tRNA genes for the expression of archaeal genes, favour semi-anaerobic conditions for the expression
of putative FMN binding domain of glutamate synthase from Methanocaldococcus jannaschii at low inducer concentrations. 相似文献
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Julie E. Croft Donald R. Love Peter L. Bergquist 《Molecular & general genetics : MGG》1987,210(3):490-497
Summary The organisation of the leucine genes in Thermus thermophilus HB8 was analysed by examining the ability of recombinant DNAs to complement Escherichia coli mutations. The arrangement of the genes is different from that in the mesophilic bacteria E. coli and Salmonella typhimurium. The promoter responsible for the expression of the leuB, leuC and leuD genes of Thermus HB8 in E. coli was identified. The sequence of Thermus DNA containing this promoter revealed structural similarities to the promoter and attenuator regions of the E. coli leucine operon. 相似文献
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Albrecht Ludwig Claudia Tengel Susanne Bauer Andreas Bubert Roland Benz Hans-Joachim Mollenkopf Werner Goebel 《Molecular & general genetics : MGG》1995,249(5):474-486
A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the over-expressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested. 相似文献