Effects of Different Preservation Methods on Activity of Recombinant E. coli

[ Objective] To explore different preservation methods of recombinant E. coli and find out the optimal conditions for preservation. [ Method] The recombinant E. coli DH5cx transformed pcDNA.3 were respectively preserved at 4℃ and -70 ℃, and the activity was determined after dif- ferent time. [ Result] The number of living E. coll with high dilutions preserved at 4 ℃ was gradually increased within the first 7 d, peaked on Day 7, and then gradually decreased. The number of living E. coli, which were preserved in 8% glycerol at -70℃ when OD800 at 0.8, were significantly higher than that of other groups after different preservation time. [ Conclusion] The optimal storage time was 7 d for recombinant E. coli at 4 ℃. For preservation at -70 ℃, the bacteria should be in logarithmic growth phase and preserved in 8% glycerol.

Exploring the modulatory role of bovine lactoferrin on the microbiome and the immune response in healthy and Shiga toxin‑producing E.coli challenged weaned piglets

Background Post-weaned piglets suffer from F18+Escherichia coli(E.coli)infections resulting in post-weaning diar-rhoea or oedema disease.Frequently used management strategies,including colistin and zinc oxide,have contrib-uted to the emergence and spread of antimicrobial resistance.Novel antimicrobials capable of directly interacting with pathogens and modulating the host immune responses are being investigated.Lactoferrin has shown promising results against porcine enterotoxigenic E.coli strains,both in vitro and in vivo.Results We investigated the influence of bovine lactoferrin(bLF)on the microbiome of healthy and infected weaned piglets.Additionally,we assessed whether bLF influenced the immune responses upon Shiga toxin-producing E.coli(STEC)infection.Therefore,2 in vivo trials were conducted:a microbiome trial and a challenge infection trial,using an F18+STEC strain.BLF did not affect theα-andβ-diversity.However,bLF groups showed a higher relative abundance(RA)for the Actinobacteria phylum and the Bifidobacterium genus in the ileal mucosa.When analysing the immune response upon infection,the STEC group exhibited a significant increase in F18-specific IgG serum levels,whereas this response was absent in the bLF group.Conclusion Taken together,the oral administration of bLF did not have a notable impact on theα-andβ-diversity of the gut microbiome in weaned piglets.Nevertheless,it did increase the RA of the Actinobacteria phylum and Bifi-dobacterium genus,which have previously been shown to play an important role in maintaining gut homeostasis.Furthermore,bLF administration during STEC infection resulted in the absence of F18-specific serum IgG responses.

Impact of an oligosaccharide-based polymer on the metabolic profiles and microbial ecology of weanling pigs experimentally infected with a pathogenic E.coli

Background Our previous study has reported that supplementation of oligosaccharide-based polymer enhances gut health and disease resistance of pigs infected with enterotoxigenic E.coli(ETEC)F18 in a manner similar to carbadox.The objective of this study was to investigate the impacts of oligosaccharide-based polymer or antibiotic on the host metabolic profiles and colon microbiota of weaned pigs experimentally infected with ETEC F18.Results Multivariate analysis highlighted the differences in the metabolic profiles of serum and colon digesta which were predominantly found between pigs supplemented with oligosaccharide-based polymer and antibiotic.The relative abundance of metabolic markers of immune responses and nutrient metabolisms,such as amino acids and carbohydrates,were significantly differentiated between the oligosaccharide-based polymer and antibiotic groups(q<0.2 and fold change>2.0).In addition,pigs in antibiotic had a reduced(P<0.05)relative abundance of Lachnospiraceae and Lactobacillaceae,whereas had greater(P<0.05)Clostridiaceae and Streptococcaceae in the colon digesta on d 11 post-inoculation(PI)compared with d 5 PI.Conclusions The impact of oligosaccharide-based polymer on the metabolic and microbial profiles of pigs is not fully understood,and further exploration is needed.However,current research suggest that various mechanisms are involved in the enhanced disease resistance and performance in ETEC-challenged pigs by supplementing this polymer.

Characteristics of β-Lactamase Synthesis in E. coli and K. pneumanie Strains in Nosocomial Infections

Background: Recently micro-organisms that synthesize extended-spectrum β-lactamase (ESBLs) were increased. The peculiarities of ESBL synthesis of Escherichia coli and Klebsiella pneumoniae strains that cause nosocomial urinary tract infections, surgical site infections and pneumonia in surgical clinic were studied. ESBL synthesis were observed 38.9% of E. coli strains obtained from urine, 92.3% of strains obtained from surgical site infections, and 50% of strains obtained from sputum. ESBL synthesis were observed 37.5% of K. pneumoniae strains obtained from urine, 85.7% of strains obtained from surgical site infections, and 60% of strains obtained from sputum. Different levels of ESBL synthesize of E. coli and K. pneumoniae strains isolated from different pattern is discussed. Conclusion. ESBL synthesis is common in E. coli and K. pneumoniae strains, which cause nosocomial infections. The frequency of occurrence of ESBL s synthesis among of these strains depends on clinical forms of nosocomial infections.

The Transport and Persistence of Escherichia coli in Leachate from Poultry Litter Amended Soils

Fecal coliform bacteria such as Escherichia coli (E. coli) are one of the main sources of groundwater pollution. An assessment of the transport and Persistence of E. coli in poultry litter amended Decatur silty Clay soil and Hartsells Sandy soil was conducted using soil columns and simulated groundwater leaching. Enumeration of initial E. coli was determined to range from 2.851 × 103 to 3.044 × 103 CFU per gram of soil. These results have been used in a batch study to determine the persistence rate of E. coli in Decatur silty Clay soil and Hartsells Sandy soil. Results prove that E. coli survival growth rate increases for clay soil later than and at a higher rate than sandy soil. The column study has determined that E. coli was transported at a rate of 3.7 × 106 CFU for Decatur silty loam and 6.3 × 106 CFU for Hartsells sandy per gram of soil. Further, linear regression analysis predictions show higher porosity and soil moisture content affect transport, and Hartsells sandy soil has higher transport of E. coli due to its higher porosity and lower volumetric water content.

Evaluating the Potential of Nitrofurantoin and Fosfomycin for E.coli UTIs: A Susceptibility Study

This study was designed to find the susceptibility of Nitrofurantoin and Fosfomycin among urinary isolates of Escherichia.coli.Four hundred(400)urine samples were collected for susceptibility of nitrofurantoin and fosfomycin among urinary isolates of E.coli.All indoor and outdoor patients'urinary samples yielded growth of E.coli.Mid-stream urine specimens were inoculated on blood agar and CLED agar and incubated at 35±2°C.Growth was observed,and Escherichia coli was identified by Gram staining,Catalase,Motility test and API 20E(Bio murex)as per standard procedure.Antimicrobial susceptibility testing of isolates for nitrofurantoin and fosfomycin was carried out by the modified Kirby-Bauer disc diffusion method according to CLSI guidelines ATCC 25922.E.coli was used as a quality control strain.A total of 400 samples were tested susceptibility of nitrofurantoin and fosfomycin among urinary isolates of E.coli during this period.A total of 400 samples yielded the growth of E.coli,out of which 178(44.5%)were male and 222(55.5%)were female samples.Among males,18(10%)were tolerant to nitrofurantoin,and 2(1.1%)were tolerant to fosfomycin.Among females,9(4.09%)were susceptible to nitrofurantoin while 6(2.72%)were susceptible to fosfomycin.Among age groups below 45 years old,6(4.76%)were tolerant to nitrofurantoin,and 2(1.58%)were sensitive to fosfomycin.Between 46-66 years old,4(2.81%)were sensitive to nitrofurantoin,and 3(2.11%)were sensitive to fosfomycin.Between 67-90 years old,17(12.87%)were sensitive to nitrofurantoin,and 4(3.03%)were tolerant to fosfomycin.Fosfomycin and nitrofurantoin showed good susceptibility in urinary isolates of E.coli and can be used empirically in our setup.

禽致病性大肠杆菌HlyE蛋白的免疫原性研究

为评估禽致病性大肠杆菌(APEC)溶血素HlyE蛋白的免疫原性,本研究对APEC溶血素HlyE蛋白进行原核表达和纯化,并对表达的重组蛋白进行SDS-PAGE和western blot分析,将纯化蛋白利用透析袋在4℃透析后,利用血琼脂平板对其溶血活性进行检测。SDS-PAGE和western blot结果显示,表达并纯化到分子量约为36 ku的重组HlyE蛋白(rHlyE),经ND-2000超微量核酸蛋白测定仪测定纯化后蛋白浓度为0.65 mg/mL;溶血活性检测结果显示,rHlyE具有溶血活性。以透析后的rHlyE作为抗原,按照50μg/只的剂量免疫小鼠,对照组于相同时间点注射等量PBS,共免疫3次间隔14 d,并于首免后不同时间采血,采用间接ELISA方法检测两组小鼠血清特异性抗体水平,并于三免后18 d以2 LD_(50)的APEC菌液攻毒小鼠,观察7 d内小鼠的死亡情况;于首免后28 d剖杀各组小鼠取其脾脏制备脾淋巴细胞,采用流式细胞术分别检测CD3^(+)、CD4^(+)、CD8^(+)T淋巴细胞亚型比率,对rHlyE的免疫原性进行评估。间接ELISA检测结果显示,该蛋白能够诱导机体产生体液免疫应答,分泌高表达量的IgG抗体,抗体水平于三免后15 d达到最高水平。rHlyE免疫攻毒保护试验结果显示,免疫组小鼠基本无明显临床症状,7 d内存活率达80%;而对照组小鼠表现明显临床症状,于攻毒后3 d内全部死亡。流式细胞术结果显示,与对照组相比,免疫组小鼠的CD3^(+)、CD4^(+)和CD8^(+)T淋巴细胞亚型比率均升高。上述结果表明,rHlyE在大肠杆菌BL21中为部分可溶性表达,将其免疫小鼠后可诱导小鼠产生较高水平的体液免疫应答,并且可对小鼠产生较好的免疫保护效果。本研究明确了APEC HlyE蛋白的免疫原性,为APEC免疫保护蛋白的筛选以及疫苗研发提供了借鉴与参考。

Recombinant E.coli LLO/OVA Vaccination Effectively Inhibits Murine Melanoma Metastasis to Lung by CD8^+T Cells Immunity

Objective： To construct recombinant E.coli LLO/OVA and investigate its tumor metastatic inhibition effect in B16 OVA melanoma challenged mice. Methods： Recombinant E.coli LLO/OVA was constructed and the expression of listeriolysin O （LLO） and ovalbumin （OVA） of the vaccine was determined by coomassie brilliant blue staining and western blotting, After 3 subcutaneous injections of E.coli LLO/OVA, the percentages of CD3^＋CD4^＋T, CD4^＋CD25^＋T, CD3^CD8^＋T and OVA257-264 SIINFEKL specific CD8^＋T cells were determined by flow cytomytry, and the tumor metastatic inhibition effect in B16 OVA melanoma challenged mice was observed. Results： Recombinant E.coli LLO/OVA was successfully constructed, and the expression of LLO and OVA of the vaccine was confirmed. After 3 subcutaneous injections of E.coli LLO/OVA and E.coli OVA in mice, the percentages of CD3^＋CD4^＋T, CD4^＋CD25^＋T and CD3^＋CD8^＋T cells were equivalent in the two groups of mice. However, there were significantly more OVA257-264 SIINFEKL specific CD8^＋T cells in E.coli LLO/OVA vaccinated mice than that in E.coli OVA vaccinated mice. The prophylactic E.coli LLO/OVA vaccination effectively prevented the tumor metastasis to lungs in B16 OVA melanoma challenged mice. Depletion of CD8^＋T cells significantly impaired the tumor inhibition effect of the vaccine in B16 OVA challenged mice. The therapeutic vaccination of E.coli LLO/OVA significantly prevented melanoma metastasis to lungs in B I6 OVA challenged mice too. Conclusion： Recombinant E.coli LLO/OVA vaccination is highly effective in inhibiting murine malignant melanoma metastasis by promoting CD8^＋T cell immunity.

Evidence and Potential Antibacterial Mechanism of Chinese Traditional Medicine Compounds for the Development of E. coli

Astragalus membranaceus (huangqi), Allium sativum (garlic), Cinnamomum cassia (cinnamon), and Dolichos lablab L. (white hyacinth bean) are the traditional Chinese herbs that were used in prescriptions in treating diarrhea caused by bacterial infection. These herbs are relatively safe for use and investigation. This study aimed to investigate the effects of Astragalus membranaceus, Allium sativum, Cinnamomum cassia, and Dolichos lablab L. on the metabolism of Escherichia coli (E. coli). The growth rate of E. coli was monitored under the influence of each herb, revealing that Astragalus membranaceus and Allium sativum exhibited significant antibacterial activity, whereas Cinnamomum cassia and Dolichos lablab L. demonstrated moderate inhibitory effects on E. coli growth. Further inhibition zone testing allowed for the evaluation of each herb’s potency and the number of generations required for E. coli to develop resistance. Additionally, the impact of the four herbs on the expression of outer membrane protein A (OmpA) in E. coli was examined by using qPCR. The findings revealed that Astragalus membranaceus acted as a sustainable bactericide by inhibiting the growth and metabolism of E. coli MG1655 through the suppression of OmpA expression. These results suggest that Astragalus membranaceus has potential as a natural antimicrobial agent for treating E. coli infections.

Genotyping Characteristics of Human Fecal Escherichia coli and Their Association with Multidrug Resistance in Miyun District, Beijing

Objective To explore the genotyping characteristics of human fecal Escherichia coli(E. coli) and the relationships between antibiotic resistance genes(ARGs) and multidrug resistance(MDR) of E. coli in Miyun District, Beijing, an area with high incidence of infectious diarrheal cases but no related data.Methods Over a period of 3 years, 94 E. coli strains were isolated from fecal samples collected from Miyun District Hospital, a surveillance hospital of the National Pathogen Identification Network. The antibiotic susceptibility of the isolates was determined by the broth microdilution method. ARGs,multilocus sequence typing(MLST), and polymorphism trees were analyzed using whole-genome sequencing data(WGS).Results This study revealed that 68.09% of the isolates had MDR, prevalent and distributed in different clades, with a relatively high rate and low pathogenicity. There was no difference in MDR between the diarrheal(49/70) and healthy groups(15/24).Conclusion We developed a random forest(RF) prediction model of TEM.1 + baeR + mphA + mphB +QnrS1 + AAC.3-IId to identify MDR status, highlighting its potential for early resistance identification. The causes of MDR are likely mobile units transmitting the ARGs. In the future, we will continue to strengthen the monitoring of ARGs and MDR, and increase the number of strains to further verify the accuracy of the MDR markers.

Escherichia coli Pathotypes in Children with Acute Diarrhea in an Informal Settlement in Nairobi, Kenya

Diarrhea is among the leading causes of morbidity and mortality in children aged Escherichia coli (DEC) accounts for 30% - 40% of childhood diarrhea cases. To identify the pathotypes involved in diarrheal outbreaks in Kenya, we analyzed archived E. coli isolates from children E. coli confirmation and antimicrobial susceptibility testing were done using the VITEK®2 instrument. Pathotype identification was performed via conventional polymerase chain reaction. Of 175 E. coli isolates, 48 (27%) were DEC pathotypes, with enteroaggregative E. coli (EAEC) predominating (71%, 34/48). Enterohemorrhagic (EHEC) and enteropathogenic E. coli (EPEC) represented 19% and 10% of isolates, respectively. Enteroinvasive and enterotoxigenic pathotypes were not identified. All DEC isolates were susceptible to amikacin, ertapenem, imipenem, meropenem and tigecycline. Conversely, most (>80%) isolates were resistant to ampicillin, ampicillin-sulbactam and sulfamethoxazole-trimethoprim. Half of all EAEC and EPEC strains were resistant to cefazolin while half of EHEC isolates were resistant to ciprofloxacin and moxifloxacin. In total, 18 resistance phenotypes were identified with “ampicillin-cefazolin-ampicillin/ sulbactam-sulfamethoxazole/trimethoprim” predominating (33%, 16/48). The majority (81%) of DEC isolates were multidrug-resistant, with extended-spectrum beta-lactamase production identified in 8% of these isolates. This study highlights the predominance of Enteroaggregative E. coli and multidrug resistance of DEC pathotypes. Studying the epidemiology of diarrheal disease and antimicrobial resistance surveillance, will aid in identifying dominant etiological agents of diarrhea and newly emerging resistant strains in informal settlements.

Establishment of Cell Free Conversion System With Biotin-labelled Recombinant PrP^(sen) Expressed in E.coli

Objective To report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope. Methods A hamster PrP protein （HaPrP） was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrP^sen preparation from scrapie strain 263K. Results Protease-resistant bands were detected after four-day incubation. Conclusion The new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.

2种氟喹诺酮类抗生素与群体感应抑制剂对E.coli的联合毒性效应

抗生素滥用带来严重的细菌耐药性,威胁生态环境和人体健康。群体感应抑制剂(quorum sensing inhibitors,QSIs)作为一种理论上难以引发细菌耐药性的新型潜在抗生素替代品,被建议单独使用或与传统抗生素联合使用。因此,考察抗生素与QSIs联合作用效应及其作用机理对其在环境中可能产生的联合暴露风险评估具有重要的参考意义。以应用较广泛的2种氟喹诺酮类药物氧氟沙星(ofloxacin,OFL)、左氧氟沙星(levofloxacin,LEV)和1种新型抗菌剂群体感应抑制剂4-羟基-2,5-二甲基-3(2H)呋喃酮(4-hydroxy-2,5-dimethyl-3(2H)-furanone,HDMF)为研究对象,运用直接均分法和均匀设计射线法分别设计3个二元和1个三元混合物体系,每个体系包含5条具有不同组分浓度比的射线。应用时间毒性微板分析法测定3种药物及其混合物体系对大肠杆菌(Escherichia coli,E.coli)的毒性,应用拟合归零法分析混合物的毒性相互作用及相互作用强度,采用分子间对接技术来探讨可能存在的作用机理。结果表明,HDMF、OFL、LEV对E.coli均具有浓度、时间依赖毒性,以半数效应浓度负对数为毒性指标,3种药物在同一暴露时间毒性顺序:LEV>OFL>HDMF。3种药物的二元混合物体系相互作用类型有拮抗/协同作用,而三元混合物体系的作用类型为协同作用,且作用类型和强度受混合物组分、暴露时间和浓度影响。氟喹诺酮类药物混合物体系中,因药物竞争结合蛋白点位而呈现出拮抗作用。在氟喹诺酮类药物和QSIs混合体系中,QSIs会破坏细菌的生物膜,使氟喹诺酮类药物更容易接触细菌造成损伤,从而呈现协同作用。但随着暴露时间的延长,氟喹诺酮类药物会对DNA造成损伤进而减少QSIs作用蛋白的产生,呈现出拮抗作用。

Detection of aac(3)IIc, aac(6)Ib, armA Genes Coding for Escherichia coli Resistance to Aminoglycosides in Burkina Faso

Background and Prupose: Antibiotic resistance is a major global health concern. In addition to the existing data on the prevalence of bacterial resistance to antibiotics, there are patchy data on bacterial resistance to aminoglycosides in Burkina Faso. In this study, we determined the prevalence of aminoglycoside resistance genes in E. coli, including aac(3)-IIc, aac(6)-Ib and armA in Ouagadougou, and determined which antibiotics in this class are most affected by resistance. Material and Methods: This study was conducted on 216 E. coli strains collected from the biomedical analysis laboratories of Saint Camille and Schiphra hospitals. E. coli strains were isolated from pus and urine samples collected between September 2018 and January 2019. Antibiotic susceptibility testing was performed using aminoglycosides, β-lactams, fluoroquinolones, and sulfonamides. Aminoglycoside resistance genes were detected in strains with at least one aminoglycoside resistance gene using conventional/multiplex PCR. Results: Aminoglycoside resistance was observed in 46.8% (101/216) of strains. The resistance rates were respectively 45.37% for Tobramycin, 32.40% for Gentamicin, 14.81% for Kanamycin, 2.31% for Netilmicin, 1.84% for Neomycin, and 0.46% for Amikacin. PCR showed that 86 strains (85.15%) possessed the aac(3)-IIc gene, 71 strains or 70.30%) possessed the aac(6’)-Ib gene, and nine strains (8.91%) possessed the armA gene. Conclusion: Aminoglycoside resistance in pathogenic E. coli strains is mainly due to the presence of the aac(3’)-IIc and aac(6’)-Ib genes. The presence of armA was first reported in Burkina Faso. Netilmicin, Neomycin and Amikacin are good therapeutic options for treating urinary tract and pus-forming infections.

Recombinant E.coli LLO/OVA Induces Murine BMDCs Maturation via TLR4 and NOD1 Receptor and Promotes Specific Cytotoxic T Cell Immunity

Objective To explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells （BMDCs） and T lymphocytes in vitro.Methods After BMDCs stimulated by E.coli LLO/OVA,their Toll-like receptor （TLR） and nucleotide-binding oligomerization domain （NOD） receptor signalling pathway were examined by superarray hybridization;and the priming effect of the vaccine activated BMDCs on CD4＋T and CD8＋T was determined by [3H]thymidine uptake and ELISA,the tumor cytotoxic effect of activated CD8＋T cells was determined by cytotoxic assay.Results After BMDCs were activated by E.coli LLO/OVA via TLR4,NOD1 receptor and NF-κB signalling pathway,the expression of their surface molecules including MHC class Ⅰ,MHC class Ⅱ,CD40,CD80 and CD86 significantly up-regulated;the secretion of IL-12 and IFN-？ increased also.The mature BMDCs stimulated the allergic CD4＋T and CD8＋T cells proliferation and their IL-2 and IFN-γ secretion,and the activated CD8＋T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.Conclusion E.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.

Performance Parameters:Demobilization Antibiotic Resistant Bacteria(ARB)and Carrying Genes(ARG)in Wastewater Disinfection

The UV irradiation is used for removing Antibiotic Resistant Bacteria(ARB)and Antibiotic Resistance Genes(ARG)from wastewater treatment.Bacteriophages are viruses that infect within bacteria,are recognized for bacterial control.The influence of some parameters in quantification and performance influencing of pathogen demobilization could be considered in disinfection of wastewater.The comparison of Polyvalent phage(NE1)versus Coliphage(NE4)in suppressing a bacterium Escherichia coli(NDM-1:b-lactam-resistant)with UV irradiation was observed the efficacy in reduction of cells in the disinfection and parameter process.The results with the effect of UV-C irradiation on NDM-1 infected with 1%of NE4 showed a decrease of cells from 8×10^(6)to 2×10^(5)in 60 min with UV-C dose.The NDM1(E.coli)was infected with 1%of NE4(Polyvalent Phage)under magnetic stirring for 1 h,the cells count was 8×10^(6).After 1 h in UV-C e×posure,the cells number reached 3×10^(5).The NDM1 that was e×posed in 1 h of UV-C irradiation and then was infected with 1%of NE4.Cells counting were done 24 h after this procedure.These cells were e×posed in UV-C and showed a reduction in the number of cells from 1×10^(8)to 4×10^(5)after 60 min.The results indicate that bacteriophages can mitigate bacteria species,and combined the conventional water disinfection technologies that can support the microbial safety control strategies.

Structure-activity correlationship and folding of recombinant Escherichia coli dihydro folate reductase (DHFR) enzyme through biochemical and biophysical approaches

The design of any antagonist or inhibitor for any enzyme requires the knowledge of structure- function relationship of the protein and the optimum conformational states for maximum and minimum activities. Furthermore, designing of the inhibitors or drugs against an enzyme becomes easier if there is information available about various well characterized intermediate conformation of the molecule. In vivo folding pathway of any recombinant protein is an important parameter for understanding its ability to fold by itself inside the cell, which always dictates the downstream processing for the purification. In the present manuscript we have discussed about the in vivo and in vitro folding, and structure-function relationship of Dihydrofolate reductase enzyme. This is an important enzyme involved in the cell growth and hence inhibition or inactivation of the enzyme may reduce the cell growth. It was observed that the equilibrium unfolding transition of DHFR proceeds through the formation of intermediates having higher exposed surface hydrophobicity, unchanged enzymatic activity and minimum changes in the secondary structural elements. Because of enhanced surface hydrophobicity, and unchanged enzymatic activity, these intermediates could be a nice target for designing drugs against DHFR.

Expression and purification of recombinant human hemangiopoietin in Escherichia coli

Objective:To express the soluble recombinant hemangiopoietin protein in E.coli BL21(DE3).Methods:Using human fetal live cDNA as a template,a partial cDNA fragment of HAPO coding N-terminal region was subcloned into plasmids pTrc99,pQE60 and pET32c to construct different recombinant prokaryotic expression systems.After selecting,the soluble rhHAPO fusion protein was expressed stably in E.coli BL21(DE3) by vector pET32c-HAPO and further isolated by nickelnitrilotriacetic acid(NTA) affinity chromatography.After cleavage with enterokinase,the rhHAPO protein was applied to Fast Flow SP sepharose column.Results:The rhHAPO protein had a purity of more than 95% and a good bioactivity based on the cell adhesion assay in ECV304 cells.Conclusion:We have established a protein engineering system to produce rhHAPO which may provide the possibility for clinical application.

重组E.coli工程菌高密度培养生产人源型胶原蛋白

Several technical parameters were studied during the fermentation of recombinant E.coli for the production of collagen-like biopolymer.The effects of dissolved oxygen as well as glucose concentration on fermentation were observed.The OD 600 value could reach 98 when dissolved oxygen was controlled at 50% and glucose around 1%.The production of human-like collagen with a yield of 29.4% was obtained.

中药三黄汤、小檗碱对E.coli生长抑制作用与庆大霉素的比较

庆大霉素对E .coli的抑菌作用强 ,试管稀释法、琼脂稀释法和平板抑菌圈法抑菌试验得到一致的MIC(3～5 μg/mL) ;三黄汤、小檗碱抑菌作用弱而稳定 ,试管稀释法测得的MIC分别为 5 0 0mg/mL和 2 .5mg/mL ,而用平板抑菌圈法得不到明显的抑菌圈 .在亚MIC药物液体分批培养时 ,庆大霉素抑菌曲线起初 2h迅速下降 ,而后回升到初始浓度 ,而三黄汤抑菌曲线平缓稳定 ,说明中药和抗生素的抑菌机制明显不同 .庆大霉素与小檗碱混合使用时 ,与庆大霉素单独使用相近 ,而与三黄混合使用时 ,则主要表现三黄汤的抑菌作用 .因此中西药结合时需要慎用 .在接近MIC药物培养基中连续传代 2 0次后 ,庆大霉素MIC提高 8倍多 ,而三黄汤和小檗碱MIC无显著变化 ,无抗药性产生 ,也不产生对庆大霉素的交叉抗药性 .研究结果还表明 ,在 1/ 4MIC三黄汤中连续传代 2 0次 ,能消除E .coli已形成的庆大霉素抗性 ,这为解决抗生素抗药性提供了线索 .图 4参 2