A three-dimensional, magnetic and electroactive nanoprobe for amperometric determination of tumor biomarkers

A novel electrochemical immunosensor for tumor biomarker detection based on three-dimensional, magnetic and electroactive nanoprobes was developed in this study. To fabricate the nanoprobes, negatively charged Fe(3)O(4) nanoparticles (Fe(3)O(4) NPs) and gold nanoparticles (Au NPs) were first loaded on the surface of multiple wall carbon nanotubes (MCNTs) which were functioned with redox-active hemin and cationic polyelectrolyte poly(dimethyldiallylammonium chloride) (PDDA). Using alpha fetoprotein (AFP) as a model analyte, AFP antibody (anti-AFP) was absorbed on the surface of Au NPs, bovine serum albumin (BSA) was then used to block sites against non-specific binding, and finally formed anti-AFP/Au NPs/Fe(3)O(4)/hemin/MCNTs named anti-AFP nanoprobes. When the target antigen AFP was present, it interacted with anti-AFP and formed an antigen-antibody complex on the nanoprobe interface. This resulted in a decreased electrochemical signal of hemin for quantitative determination of AFP when immobilized onto the screen-printed working electrode (SPCE). The results showed that the nanoprobe-based electrochemical immunosensor was sensitive to AFP detection at a concentration of 0.1 to 200 ng·mL(-1) with a detection limit of 0.04 ng·mL(-1), it also demonstrated good selectivity against other interferential substances. The electroactive nanoprobes can be massively prepared, easily immobilized on the SPCE for target detection and rapidly renewed with a magnet. The proposed immunosensor is fast, simple, sensitive, stable, magnet-controlled, nontoxic, label-free and reproducible.     

泌尿道致病性大肠埃希菌PapG重组蛋白对小鼠尿道上行感染的免疫保护作用

目的探讨泌尿道致病性大肠埃希菌(UPEC)P菌毛粘附素PapG重组融合蛋白对小鼠尿道上行感染的免疫保护作用。方法纯化蛋白按一定程序免疫BALB/c雌性小鼠,末次免疫后第7天用UPEC临床分离株进行尿道上行攻击;攻击后第5天检测尿液和肾脏培养菌,同时测定血清抗体效价。结果经GST-PapG重组蛋白和GST纯化蛋白免疫的小鼠体内均产生了相应的抗体,而仅经重组蛋白免疫的小鼠具有一定的抵抗毒株上行感染的能力,表现为肾脏培养菌量均明显减少。结论PapG粘附素与GST构成的重组融合蛋白对小鼠尿道上行感染具有一定的免疫保护作用。     

牛源大肠杆菌F41菌毛蛋白的原核表达和多克隆抗体的制备

目的原核表达及纯化牛源产肠毒素性大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)F41菌毛蛋白,并制备F41菌毛蛋白的多克隆抗体。方法以ETEC基因组为模板,采用PCR法扩增F41菌毛基因,克隆入表达载体pQE-30,转化大肠杆菌XL1-Blue,IPTG诱导表达,并进行SDS-PAGE分析,表达产物经切胶纯化,免疫新西兰大白兔,制备多克隆抗体,采用间接ELISA法测定抗血清效价,Western blot分析F41菌毛蛋白与多抗的反应原性。结果重组表达质粒pQE-30-F41经双酶切及测序证实构建正确;F41菌毛重组蛋白以包涵体形式表达,相对分子质量约为32 000;重组蛋白的表达量约占菌体总蛋白的35%,纯化的重组蛋白纯度可达92%;制备的抗血清效价达1∶2.56×106以上,Western blot结果表明F41菌毛蛋白与制备的多抗具有良好的反应原性。结论已成功原核表达并纯化了F41菌毛重组蛋白,且制备了高效价的多克隆抗体,为单克隆抗体制备及其免疫检测方法的建立奠定了基础。     

基于pH恒定补糖的生物基丁二酸高效合成

通过确定大肠杆菌在产酸阶段葡萄糖的消耗与酸中和剂碳酸钠间的定量关系,建立了pH恒定补糖策略,能够使发酵液中葡萄糖浓度维持在稳定水平。与分批发酵相比,采用pH恒定补糖且维持葡萄糖浓度在较低的水平对丁二酸的积累是有利的。当采用pH恒定补糖并控制葡萄糖质量浓度在10g/L时,丁二酸最终质量浓度达到57.6 g/L,生产效率达到1.15 g/(L·h)。     

INVITED REVIEW ON THE USE OF CHEMICALLY STRUCTURED MODELS FOR BIOREACTORS

An individual cell is an immensely complicated self-regulated chemical reactor that can alter its biosynthetic machinery to meet the demands of a changing environment. The biochemical engineer must build a large macroscopic reactor to harness the cells for desirable chemical conversions. The design and control of such bioreactors would be facilitated with effective mathematical models of the response of the culture to changes in nutrients or other environmental variables. Because of the inherent internal plasticity of the cell, models must reflect the changing structure of the biomass. This paper reviews some examples of models which contain components representing various chemical fractions within the cell. The advantage of these models is their potential ability to predict the dynamic behavior of a cellular population. In addition such models are potential tools for testing hypotheses concerning cellular control mechanisms and consequently the development of more effective cell strains. Models of populations based on a finite-representation technique using an ensemble of chemically structured single-cell models are emphasized. These latter models are capable of accurate a priori prediction of bioreactors to perturbations in flow rates or feed concentrations. Models which aspire to the a priori quantitative prediction of cell population behavior must be sufficiently complex that shifts in growth-rate limiting processes can be taken into account; consequently a high-level of chemical structure will characterize the best models.     

大肠杆菌生产琥珀酸的代谢工程研究进展

琥珀酸是一种重要的化工原料,具有广阔的市场. 微生物发酵法生产琥珀酸可以解决常规化学合成法对石油的依赖. 代谢工程的兴起使重组大肠杆菌生产琥珀酸变为可能,也取得了一定的成效,但是其发酵强度还不够高,且过程中伴随着其他有机酸的积累,因此还不适于工业化生产. 代谢工程以系统生物学为基础,为重组大肠杆菌的进一步改造提供了更合理的依据. 本工作以大肠杆菌生产琥珀酸所涉及的关键酶、代谢途径及其改造为对象,系统综述了大肠杆菌生产琥珀酸所涉及的代谢工程技术及其最新研究进展,并探讨了将来的发展前景.     

外源蛋白在大肠杆菌中的可溶性表达策略

长期以来,大肠杆菌一直是表达外源蛋白的首选表达系统. 但由于外源蛋白在表达过程中容易被宿主细胞蛋白酶降解或者形成包涵体,其应用受到了限制. 本文综述了在大肠杆菌中表达可溶外源蛋白的策略和进展,以期提高具有生物活性的外源基因的表达水平.     

A MICROCALORIMETRIC METHOD TO STUDY THE EFFECT OF KANAMYCIN ON THE METABOLISM OF RECOMBINANT ESCHERICHIA COLI

A microcalorimetric technique based on the metabolic heat production from cultured cells was used to investigate the effect of kanamycin on the growth and metabolism of recombinant Escherichia coli. Power-time curves of growing recombinant Escherichia coli cell suspensions treated with different kanamycin doses were recorded and were described very accurately by the generalized logistic equation. The rate constant (k) is kanamycin concentration-dependent and the relationship between k and C (kanamycin dose) is nearly linear. The 50% inhibitory concentration IC₅₀ was 88.49 µg/mL. The experimental results revealed that high doses of kanamycin inhibited the growth of recombinant E. coli during the log phase and promoted growth during the stationary phase. These results are important for deciding the optimum kanamycin dose for the maximal synthesis of human-like collagen.     

Red同源重组在大肠杆菌基因敲除中的应用

大肠杆菌基因敲除技术发展迅速,各种新型技术的出现使得其基因组的改造较以往更为快速、简单,尤其是Red同源重组技术在大肠杆菌基因敲除中的应用已经越来越成熟。综述了几种不同的Red同源重组技术,并分析了每种方法的原理及操作策略,指出了各种方法的特点及优势。     

Antibacterial Mechanism of Octamethylene-1,8-Bis(Dodecyldimethylammonium Bromide) Against <Emphasis Type="Italic">E. coli</Emphasis>

The antibacterial activity of the cationic dimeric amphiphile octamethylene-1,8-bis(dodecyldimethylammonium bromide) (12-8-12) against the Gram-negative bacteria Escherichia coli was measured and compared with the monomeric amphiphile dodecyltrimethylammonium bromide (DTAB). The minimum inhibitory concentration (MIC) of 12-8-12 was about 1/20 of DTAB. By measuring the activity of β-galactosidase and the image of field-emission scanning electron microscopy, it was revealed that the cationic amphiphile 12-8-12 interacted with the negatively charged membrane of E. coli and disrupted the membrane integrity, thus leading to the release of intracellular contents and the death of bacteria. To further explore the antibacterial mechanism, the interactions of cationic amphiphile 12-8-12 with biomacromolecules (bovine serum albumin (BSA) and salmon sperm DNA) were studied by measuring the intrinsic fluorescence of BSA and the zeta potential of DNA. It was shown that the antibacterial action site of cationic amphiphile was not only on the bacterial membrane but also on the intracellular contents such as protein and DNA.     

运用试验读者设计方法提高胞外杂合β-葡聚糖酶在重组大肠杆菌中的表达

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid extracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L-1. After optimization of the medium, 297.71U·ml-1 of β-glucanase activity in the medium and 352350U·g-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those obtained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.     

大肠埃希菌和肺炎克雷伯菌临床分离株的耐药性分析

目的分析临床分离的大肠埃希菌和肺炎克雷伯菌的分布情况及耐药性。方法收集吉林大学中日联谊医院临床标本中分离的221株大肠埃希菌和152株肺炎克雷伯菌,采用纸片扩散法检测抗菌素的敏感性;双纸片协同试验和纸片表型确证试验筛选并确证产超广谱β-内酰胺酶的菌株;按照美国国家临床实验室标准化委员会(NCCLS)2005年版的标准判断结果。结果大肠埃希菌和肺炎克雷伯菌对亚胺培南和美罗培南的敏感性最高,均达100%;对头孢他啶、哌拉西林/他唑巴坦和头孢西丁的敏感性也较高,均大于70%,而对氨苄西林的耐药性均大于90%。共检出产超广谱β-内酰胺酶的大肠埃希菌118株,检出率为53.4%;肺炎克雷伯菌21株,检出率为13.8%;产超广谱β-内酰胺酶的两种菌株与非产超广谱β-内酰胺酶的同种菌株相比,对抗菌素的耐药性均明显增加。结论大肠埃希菌和肺炎克雷伯菌仍是产超广谱的β-内酰胺酶的主要菌株,且对常用抗菌素产生了较高的耐药性。     

L-天冬氨酸固定床列管反应器的实验研究

本文对固定化大肠杆菌连续生产L-天冬氨酸的管式固定床反应器进行了实验研究。结果表明：固定床反应器能够有效地排除由富马酸和氨生成L-天冬氨酸的反应热．在底物浓度为1．0mol／L的条件下，循环冷却水的温度为37℃、废物在反应器内的停留时间为2～6h（即空速低于0．5／h）时，由富马酸生成L-天冬氨酸的转化率高于99，见反应器内的最高温度不超过37．3℃。     

重组大肠杆菌共表达D-海因酶和N-氨甲酰基-D-氨基酸酰胺水解酶

通过PCR分别扩增得到N-carbamoylase基因和上游带有RBS区的D-hydantoinase基因,并将其依次克隆入pBAD/His A质粒中,得到呈多顺反子结构的双酶表达质粒pBAD-CRH,转化E. coli Top10F￠得到重组菌TaraCRH. 实验证明,E. coli TaraCRH对外源蛋白的表达控制严紧,而加入阿拉伯糖诱导后可以成功表达两酶基因. 诱导条件优化结果表明,诱导温度采用29℃,培养基中阿拉伯糖浓度0.05 g/L可达到最高酶活. 进一步尝试采用玉米浆培养基代替LB培养基,在经过优化的玉米浆培养基中培养TaraCRH菌株,经阿拉伯糖诱导,海因酶和水解酶酶活分别达到3.52和4.21 U/mL.     

利用转氨反应制备L－苯丙氨酸

利用选择性培养基筛选到一株具有高转氨酶活性的大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｔ），能够高效地把苯丙酮酸和Ｌ－天冬氨酸转化成Ｌ一苯丙氨酸，利用游离细胞转化苯丙酮酸，反应６ｈ，可生成７０ｇ／ＬＬ－苯丙氨酸，转化率达９０％。利用固定化细胞转化苯丙酮酸，反应８ｈ可生成５９．６ｇ／ＬＬ－苯丙氨酸，转化率达７３％。