大肠杆菌中同时表达的可溶性和不溶性人α-干扰素-2b的初步分离研究

1 INTRODUCTION Interferons are proteins naturally occurring in the hu-man body with antiviral, antiproliferative and immu-noregulatory activity[1]. The commercial production of interferons as important clinic medicines was carried out by using recombinant E. coli system[2]. Despite the ease of cloning and genetic manipulation in E. coli, heterogeneous protein was almost expressed as inclusion body form since the outer membrane appears to be a very effective barrier to the release of pro…     

重组大肠杆菌流加发酵中短暂溶氧休克促进5-氨基乙酰丙酸生成(英文)

5-Aminolevulinic acid （ALA） is a common precursor for tetrapyrrole compounds in all kinds of organ isms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolved oxygen （DO） shock during aerobic fermentation, was introduced to produce 5-aminolevulinic acid with a recombi-nant E. coli. Effects of duration time of DO shock operation on plasmid concentration, intracellular ALA synthase （ALAS） activity and ALA production were investigated in Erlenmeyer shake flasks. The results indicated that both ALAS activity and ALA yield were enhanced in an anaerobic operation of 45 rain in the early exponential phase during fermentation, while they decreased when the anaerobic operation time was further increased to 60 rain. The DO shock protocol was confirmed with the fed-batch fermentation in a 15 L fermenter and the ALA production achieved 9.4 g.L-1 （72 mmol.L-1）, which is the highest yield in the fermentation broth reported up to now.     

补料分批培养耐乙酸大肠杆菌生产人表皮生长因子

An acetate-tolerant mutant of Escherichia coli DH5α, DA19, was used for secretory production of human epidermal growth factor （hEGF） whose expression was under the control of phoA promoter. The recombinant cells were cultured in a chemically defined medium, and glucose was added at different specific provision rates during the growth and expression phases. It was found that pH had a significant effect on the extracellular hEGF production. The extracellular hEGF concentration was 75.5mg·L^-1, 5.2-fold of the level reached at pH 7.0, even though more acetate was produced. Nitrogen source was limited in the later growth phase. Supplementation of ammonium promoted the consumption of phosphate and reduced the time to exhaust phosphate, but the extracellular hEGF production was similar to that without supplementation of ammonium.     

Preliminary Study on E. coli Microbial Fuel Cell and On-electrode Taming of the Biocatalyst

A mediator microbial fuel cell (MFC) was constructed by using E. coli as biocatalyst and new methylene blue as electron mediator. E. coli cells were carried out in anaerobic growth prior to inoculating them into the MFC in order to pre-adapt bacterial metabolism in an anaerobic environment, the electricity generation of MFC was tested, its maximum power density reached 263.94 mW/m2 with the corresponding current density 1287.50 mA/m2, the internal resistance of MFC was 200 W, and capability of the MFC was even better than those reported so far. Moreover, on-electrode taming method was adopted to improve electrochemical activity of E. coli, namely a combination of E. coli taming and electricity generation simultaneously in the same MFC without scraping off the biofilm of MFC, after the 4th on-electrode taming, the tamed E. coli MFC showed a 54% improvement in peak current density, being 612.50 mA/m2, and a 64% improvement in the maximum power output, being 166.67 mW/m2, compared with that of parental E. coli MFC. And the maturation time of tamed biofilm was obviously reduced to 240 min, quickening up 1 times compared with that of parental E. coli biofilm.     

一种通过重组大肠杆菌和重组恶臭假单胞菌生产3-羟基癸酸的新方法

3-hydroxydecanoic acid (3HD) is an interesting intermediate for chemical synthesis of many valuable compounds. A novel method to produce 3HD by recombinant bacteria was constructed in Escherichia coli HB101 and Pseudomonas putida GPp104, respectively. Simultaneous expression of both phaG encoding (R)-3-hydroxydecanoyl-ACP:CoA transacylase and tesB encoding thioesterase Ⅱ in E. coli HB101 increased 3HD production approximate 1.7-folds compared with the expression of phaG gene alone under identical conditions. In addition, when the tesB gene was introduced into the strain, the polyhydroxyalkanoate synthase negative strain P. putida GPpl04 produced extracellular 3HD. Thus, a novel pathway to produce 3HD by recombinant Pseudomonas was constructed. It was also found that the ratio of carbon source to nitrogen source affected the production of 3HD by recombinant P.putida harboring tesB gene. Nitrogen limitation seemed to promote the extracellular 3HD production.     

Simultaneous Cell Disruption and Aqueous Two-Phase Extraction for Isolation of Intracellular Recombinant Proteins

A new technique was developed for the integrated processing of cell disruption and aqueous two-phase extraction in a high-speed bead mill to separate intracellular proteins form microbial cells. The process was narned as simultanecus cell disruption and aqueous two-phase extraction (ADATE). Advantages, such as high cell disruption efficiency, biochemical activities proservation of proteins, cell debris elimination, and preliminary puriffcation of the target protein were being clairmed. When this technique was employed for isolating recombinant Tumor Necrois Factor (TNF) from E.coli, overall protein codcentration and TNF activity were found to have been increased. More than 85% of TNF was partitioned into the top phase and all cell debris were in the bottom phase. The partition coefficinet was greater than 3 and the TNF puriflcation fsctop was greater than 6. It is zhown that less separation steps were being utilized in the new techniqne, meaning a reduction in separation time and less process extractors required.     

Immobilization of urease in metal–organic frameworks via biomimetic mineralization and its application in urea degradation

Enzyme immobilization has been accepted as an efficient technique for improving the stability and recyclability of enzymes. Herein, biomimetic mineralization strategy was employed to achieve the immobilization of urease in a type of metal–organic frameworks(zeolite imidazolate framework-8, ZIF-8), and the immobilized enzyme urease@ZIF-8 was systematically evaluated for its structure, activity, stability and recyclability, using the hydrolysis of urea as a model. The entrapment of urease was found to be realized in a synchronous manner with the formation of ZIF-8 crystal. The loading of urease in ZIF-8 was measured to be ca. 10.6% through the bicinchoninic acid(BCA) protein assay. The encapsulated urease could efficiently maintain its native conformation, which endowed the immobilized urease with excellent activity and stability, even in harsh conditions(e.g., in the presence of trypsin, acidic or alkali conditions, or at high temperature). Further, urease@ZIF-8 exhibited good recyclability during the degradation of urea, in which it could keep 58.86% of initial activity after being used for 5 cycles. Thus, biomimetic mineralization could be potentially utilized as a promising method to prepare immobilized ureases with superior activity, stability and recyclability, thereby facilitating the construction of efficient catalysts for industrial biocatalysis and biosensing.     

大肠杆菌中表达来源于Agrobacterium radibacter的新hamA基因以生产5-氨基乙酰丙醇

A new hemA gene encoding 5-aminolevulinate （ALA） synthase was cloned from Agrobacterium radiobacter zju-0121. The ALA synthase catalyzes the pyridoxal phosphate-dependent condensation of succinyl coenzyme A （succinyl-CoA） and glycine to produce ALA. Four plasmids carrying the A. radiobacter hemA gene were transformed into different E. coli strains. The effects of both genetic and physiological factors on the expression of ALA synthase and ALA production were studied. The results indicated that the final intracellular activity of ALA synthase and the production of ALA in different expression systems varied largely. Among them, the recombinant E. coli BL21 （DE3） harboring the expression plasmid pET28-A. R-hemA was the most suitable one. The effects of isopropyl-β-D-thiogalactopyranoside （IPTG） addition time, IPTG concentration, culture temperature and the initial concentration of precursors and glucose on the ALA production were also evaluated. The expressed ALA synthase accounted for about 23.7% of the intracellular soluble protein. The highest specific activity of ALA syn- thase was 13.8 nmol.min^-1.mg^-1 of intracellular soluble protein. In the batch culture of the recombinant E. coli, the extracellular ALA concentration reached 0.9g.L^-1.     

Chemical characterization of lysyl oxidase inhibitor from avocado seed oil

The active factor of lysyl oxidase inhibition was separated from unsaponifiables of avocado seed oil and characterized by gas chromatography-mass spectrometry. Results indicated the presence of furan-containing lipids in the active factor mixture and also showed a structural difference compared to previously reported furan-containing lipids of avocado which relates to the length of the hydrocarbon chain substituent. Another structural difference evinced was the availability of the hydroxyl group in the aliphatic moiety of the investigated substances. A purified mixture of furan-containing compounds was testedin vitro for inhibitory activity on pure bovine aorta lysyl oxidase. It was shown that mixing furan-containing lipids in Tween 80 reversibly inhibited pure bovine aorta lysyl oxidase activity against tritiated recombinant tropoelastin with the I₅₀ value of inhibition of 105 μM. Thesein vitro studies suggested that the mixture of avocado seed oil furan-containing lipids was not a substrate-specific inhibitor of lysyl oxidase, and it might prove to be useful as a potential antifibrotic drug. Moreover, the unique chemistry of the studied compound for lysyl oxidase inhibition should enable the designing of new probes of the active site of this important enzyme.     

Synthesis and Antimicrobial Activity of Boron-doped Titania Nano-materlals

Antibacterial activity of boron-doped TiO2 （B/TiO2） nano-materials under visible light irradiation and in the dark was investigated. A simple sol-gel method was used to synthesize TiO2 nano-materials. X-ray diffraction pattern of B/TiO2 nano-materials represents the diffraction peaks relating to the crystal planes of TiO2 （anatase and rutile）. X-ray photoelectron spectroscopy result shows that part of boron ions incorporates into TiO2 lattice to form a possible chemical environment like Ti-O-B and the rest exist in the form of B2O3. The study on antibacterial effect of B/TiO2 nano-materials on fungal Candida albicans （ATCC10231）, Gram-negative Escherichia coli （ATCC25922） and Gram-positive Staphylococcus aureus （ATCC6538） shows that the antibacterial action is more significant on Candida albicans than on Escherichia coli and Staphylococcus aureus. Under visible light irradiation, the antibacterial activity is superior to that in the dark.     

HIV-1 Vif与ElonginC蛋白在大肠杆菌中的共表达

目的在大肠杆菌中共表达HIV-1Vif与ElonginC蛋白。方法PCR扩增ElonginC基因全长DNA片段,克隆入pMDT-easy载体,经NdeⅠ/BamHⅠ双酶切后,与质粒pRSETB连接,构建质粒pRSETB-ElonginC。将质粒pMRI-Vif转化大肠杆菌BL21(DE3),获得表达菌株,再将此菌株制备成感受态细胞,用pRSETB-ElonginC质粒转化该细胞,经1mmol/L IPTG诱导,表达产物经SDS-PAGE及Western blot鉴定。结果质粒pRSETB-ElonginC经NdeⅠ/BamHⅠ双酶切,可切出约3000和400bp的2条片段,测序证明质粒构建正确。在大肠杆菌中共表达了Vif与ElonginC蛋白,该蛋白具有良好的抗原特异性。结论在大肠杆菌中共表达了Vif与ElonginC蛋白。     

HIV跨膜蛋白gp36和gp41的截短及融合表达

目的将HIV-1的跨膜蛋白gp41和HIV-2跨膜蛋白gp36进行截短,并在大肠杆菌中进行融合表达。方法用PCR将gp41和gp36的编码基因进行截短,回收的PCR产物纯化后克隆到连接载体pGEM-T上,然后用BamHⅠ、EcoRⅠ和SalⅠ切下目的基因,并构建到表达载体pGEX-4T-3,导入宿主细胞BL21,用IPTG诱导表达。结果酶切鉴定显示,截短的HIV-1gp41和HIV-2gp36跨膜蛋白基因大小与预期的一致,表达产物经SDS-PAGE分析显示在相对分子质量66000处出现融合表达条带,Westernblot分析显示,与相应抗体出现特异性反应。结论已成功对gp41和gp36跨膜蛋白进行截短,并构建表达载体进行表达,为跨膜蛋白的进一步应用研究奠定基础。     

MBP融合肝素酶Ⅲ大肠杆菌高效表达体系构建

肝素酶Ⅲ（heparinase Ⅲ,HepC）是一种重要的多糖裂解酶,在抗癌药物开发、低分子量肝素生产以及肝素类药物的质量控制等方面具有重要的作用。目前制约其工业应用前景的主要技术瓶颈是生产成本高,异源重组表达效果差,缺乏高效的表达方法。本研究借鉴前期经验,通过HepC基因的密码子优化,并与麦芽糖结合蛋白（maltose-binding protein,MBP）融合,构建了MBP-coHepC（基因密码子优化后的蛋白为coHepC）的大肠杆菌表达体系,结合培养条件优化大幅度提高了HepC的可溶表达比例,其摇瓶发酵总酶活值达到7603.46 IU·L^-1;同时,本研究从转录和翻译水平揭示了HepC表达效果提高的可能原因。这些研究为肝素酶Ⅲ的应用发展提供了基础。     

犬干扰素α1基因在大肠埃希菌中的表达及其抗病毒活性的测定

目的在大肠埃希菌中高效表达重组犬干扰素α1(canine interferon alpha1,CaIFNα1)基因,并对其抗病毒活性进行初步研究。方法根据大肠埃希菌密码子的偏嗜性,人工合成犬干扰素α1成熟蛋白编码基因,同时引入大肠埃希菌EcoRⅠ和XhoⅠ酶切位点,将改造后合成的CaIFNα1核苷酸序列定向克隆至原核表达载体pET-32a(+),构建重组表达质粒pET-CaIFNα1,并进行PCR及双酶切鉴定;将鉴定正确的重组表达质粒pET-CaIFNα1转化BL21(DE3),IPTG诱导表达,表达的重组融合蛋白经SDS-PAGE及Western blot鉴定,并采用MDCK/VSV微量细胞病变抑制法检测其抗VSV病毒活性。结果重组表达质粒经PCR及双酶切鉴定,证明构建正确;重组融合蛋白CaIFNα1相对分子质量约39 000,主要以包涵体形式存在,表达量约占菌体总蛋白的61.5%,可被鼠抗His单克隆抗体及鼠抗CaIFNα1多克隆抗体特异性识别,在MDCK细胞上呈现较高的抗病毒活性,为2.0×106U/ml。结论已成功改造了CaIFNα1基因,并在大肠埃希菌中实现了高效表达,表达产物具有较高的抗病毒活性。     

小鼠载脂蛋白A-Ⅰ的原核表达及抗血清的制备

目的原核表达小鼠载脂蛋白A-Ⅰ(ApoA-Ⅰ),并制备抗血清。方法应用RT-PCR方法从小鼠肝脏中扩增ApoA-Ⅰ基因,亚克隆至原核表达载体pET-28a(+),转化大肠杆菌BL21(DE3),IPTG诱导表达。表达的融合蛋白经Ni2+-NTA柱纯化后,免疫家兔,制备抗血清。结果扩增的ApoA-Ⅰ基因经测序,表明与GenBank中报道的序列(NM_009692)完全相同。重组表达质粒经酶切鉴定,证明构建正确。表达的ApoA-Ⅰ融合蛋白相对分子质量约为32000,表达量占菌体总蛋白的17%。纯化的融合蛋白纯度达90.7%,可与抗His·Tag单抗发生特异性反应。制备的抗血清可识别ApoA-Ⅰ融合蛋白,效价可达1∶105。结论已在大肠杆菌中表达了小鼠ApoA-Ⅰ,并制备了较高效价的抗血清。     

重组人白细胞介素-10在大肠杆菌中的表达及生物学活性分析

目的在大肠杆菌中表达重组人白细胞介素-10(Interleukin-10,IL-10),并检测其生物学活性。方法将IL-10基因重组到质粒pET11c中,转化BL21(DE),提取质粒,经酶切鉴定和测序分析;在25℃用低浓度的IPTG诱导表达,对包涵体IL-10稀释复性;经ELISA检测其含量,MC/9细胞增殖法检测其生物学活性。结果工程菌IL-10/pET11c/BL21诱导表达的目的蛋白以可溶性和包涵体两种形式存在,Westernblot鉴定证实为IL-10蛋白,两种形式的IL-10均具有一定的生物学活性。结论已成功地在大肠杆菌中表达了IL-10,为进一步纯化和制备IL-10的基因工程药物打下了基础。     

蜂毒肽/变构hIL-2融合蛋白的原核表达及其对宿主菌生长的影响

目的构建蜂毒肽(Melittin)与变构hIL-2融合基因原核表达质粒,并检测表达的融合蛋白对宿主菌E.coli生长的影响。方法以质粒pGEX-4T-2/Melittin-IL-2(88Arg)为模板,通过PCR定点诱变为Melittin-IL-2(88Arg,125Ala),将PCR产物和pET-15b载体分别经双酶切后连接,构建重组表达质粒pET-15b/M-IL-2(88Arg,125Ala),转化E.coliBL21(DE3),IPTG诱导表达。SDS-PAGE及ELISA分析表达产物;检测诱导不同时间重组菌的A600值,绘制生长曲线,并进行活菌计数。结果PCR扩增的目的片段长542bp,重组表达质粒测序分析证明目的基因如预期突变;ELISA可检测到目的蛋白表达,但表达量较低;SDS-PAGE分析未见目的条带;诱导表达4h,重组菌A600值由0.8下降至0.6;诱导2h,活菌计数由108个/ml降至104个/ml。结论已成功构建了原核表达质粒pET-15b/M-IL-2(88Arg,125Ala),表达的融合蛋白可能对宿主菌具有毒性,从而杀伤宿主菌。     

幽门螺杆菌尿素酶B亚单位基因的克隆及其高效表达

目的克隆幽门螺杆菌尿素酶B亚单位(UreB)基因,构建原核表达载体,并进行高效表达。方法以幽门螺杆菌基因组DNA为模板,PCR扩增UreB基因,双酶切后,与质粒pET-22b(+)连接,构建表达载体pET-22b(+)/UreB,分别转化E.coliBL21(DE3)、Origam(iDE3)和Rossetta(DE3),经IPTG诱导后,进行SDS-PAGE和Western blot分析。结果经酶切及测序,证明幽门螺杆菌UreB基因的原核表达载体构建正确。3种重组菌的诱导表达产物经SDS-PAGE分析,均可见相对分子质量为64000的目的蛋白条带,Rossetta(DE3)重组菌目的蛋白表达量最高,约占菌体蛋白的35%。Western blot结果表明,表达的目的蛋白具有良好的反应原性。结论已成功克隆了幽门螺杆菌UreB基因,并在大肠杆菌Rossetta(DE3)中获得了高效表达。     

柯萨奇B4病毒非结构蛋白P_2C基因的克隆与表达

目的克隆并表达柯萨奇B4病毒(CVB4)非结构蛋白P2C基因。方法提取CVB4总RNA,RT-PCR扩增P2C基因,克隆入pUCm-T载体中,进行酶切和测序鉴定。双酶切重组质粒pUCm-T-P2C,将P2C基因片段定向亚克隆至原核表达载体pMAL-C2中,转化大肠杆菌JM109,IPTG诱导表达,SDS-PAGE和Western blot鉴定表达产物。结果RT-PCR扩增得到987bp的基因片段,测序结果与GenBank公布的P2C基因序列一致。pMAL-C2-P2C经双酶切鉴定,证明构建正确。表达的融合蛋白相对分子质量约78000,表达量约占菌体总蛋白的25%,且具有良好的反应原性。结论已成功克隆并表达了CVB4P2C基因,为进一步研究其生物学活性奠定了基础。     

深黄被孢霉Δ5-脱饱和酶的原核表达及纯化

目的原核表达并纯化深黄被孢霉Δ5-脱饱和酶。方法采用RT-PCR技术扩增深黄被孢霉Δ5-脱饱和酶基因,插入表达载体pET-30a(+)中,构建重组表达质粒pET-D5D,转化入大肠埃希菌(E.coli)BL21(DE3)中,IPTG诱导表达。表达的重组蛋白经Ni2+-NTA纯化后,进行Western blot鉴定。结果重组表达质粒pET-D5D经双酶切(NotⅠ/SalⅠ)和测序鉴定,证明构建正确;表达的重组蛋白相对分子质量约为55 000,诱导6 h表达量较高,主要以包涵体形式表达;纯化的重组蛋白可与兔抗Δ5-脱饱和酶多克隆抗体特异性结合。结论成功在E.coli中表达了深黄被孢霉Δ5-脱饱和酶,纯化后的重组蛋白反应原性良好,为进一步研究Δ5-脱饱和酶的功能奠定了基础。