Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.

Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E.coli

AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli . RESULTS Sequence analysis indicated that the HCV isolate of this study belongs to HCV Ⅱ; SDS PAGE demonstrated an M r 23800 and an M r 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.

High-level expression of human calmodulin in E.coli and its effects on cell proliferation

Calmodulin (CaM), widely distributed in almost all eukaryotic cells, is a major intracellular calcium receptor responsible for mediating the Ca2 + signal to a multitude of different enzyme systems and is thought to play a vital role in the regulation of cell proliferative cycle[1，2]. Recently, many studies showed that CaM is also present in extracellular fluid such as cell culture media and normal body fluid and has been reported to stimulate proliferation in a range of normal and neoplastic cells, apparently acting as an autocrine growth factor[3-11]. In 1988, Crocker et al reported for the first time that addition of extracellular pure pig brain CaM could promote DNA synthesis and cell [7]proliferation in K562 human leukaemic lymphocytes[7].After that, more and more research was done on extracellular CaM and evidences demonstrated that extracellular CaM could also stimulate cell proliferation in normal human umbilical vein endothelial cells[5], keratinocytes[4], suspension-cultured cells of Angelica Dahurica, etc[6]. CaM is a monomeric protein of 148 amino acids that contains four homologous Ca2 + -binding domains. CaM has been highly conserved throughout the evolution. Only 1 out of 148 amino acids of human CaM is different from that of fish CaM. Complementary DNAs encoding rat, eel, chicken, human, and trypanosome CaM have been cloned.

An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.

Receptor-binding domain of SARS-Cov spike protein: Soluble expression in E.coli, purification and functional characterization

AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli

AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori(H pylori)neutrophil-activating protein (NAP) and E. coli maltosebinding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis.Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment,Western blotting with human H pylori anti-sera.RESULTS: E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG.The molecular weight of rBMP-NAP was about 57 kD,accounting for 37.55% of the total protein in the sonicated supematant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture.The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself.CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.

Expression of Core Domain of Porcine Zona Pellucida 3β in E.coli

To obtain the recombinant core domain of porcine zone pellucida 3β （cZP3β） for the further research on its functions Methods The nucleotide sequence region from 44 to 306 codons of pZP3β entire eDNA was obtained by PCR and then was cloned into pET-3c vector. After being identified, recon was transformed into E.coli BL21 （DE3） pLysS and then induced by IPTG. Results The recombinant cZP3β was expressed in E. coli up to 15% of total cellular proteins, and was made sure by Western blot analysis. Conclusion The research on expression of core domain of pZP3β could benefit to further investigation of its immunogenicity and the development of antigen preparation.

Inducible Expression and Splicing of Candida Group I Ribozyme in E.coli

The Ca. LSU intron flanking a 129 bp cxon upstream and a fOO bp exondownstream was inserted into the lacZ gene on pRS426 to transform E. coli. Northern blot analysisand RT-PCR showed that splicing of Ca. LSU in E. coli is efficient upon inducible expression of theprecursor RNA, In contrast, co-lranscriptional self-splicing of the intron in vitro is much lessactive. Therefore, this E. coli splicing system can be used as a better model to investigate theeffect of the ribozyme inhibitors on Ca. LSUsplicing in living cell. We examined (he effects ofneomycin sulfatc and pentamidine on Ca. LSU splicing in E. coli, and found that these drugsdoes-dependently inhibit the intron splicing. However, heomycin is more potent than pentamidine inthis action.

Cloning and high-level expression of human interferon alpha-8 in E.coli

Human interferon alpha-8(IFN-α8) is an important cytokine with multiple biological functions.A genetically engineered strain, E. coli XL1-Blue/pBm, was constructed by DNA recombination technology and characterized by restriction analysis, DNA sequencing.

Expression of the human era cDNA in E.coli

Objective: To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX-4T3 in which exogenous gene was controlled by Ptac promoter. The recombinant plasmid pGEX-Hera was transformed into DH5 (and induced with IPTG chemically. Results: The human era gene was amplified and the sequence was correct. When the bacteria with pGEX-Hera was induced, an anticipated 65 000 protein band appeared on SDS-PAGE gel and amounted to 23% of total bacterial protein. Conclusion: The human era gene has been successfully amplified and efficiently expressed in E.coli.

Differential expression of bcl-2 and bax genes during the E.coli-induced apoptosis of human U937 cells

To explore the role of bcl-2 and bax genes in the apoptosis of human U937 cells induced by E.coli, flow cytometry assay with annexinⅤ-FITC/PI double staining was used to determine the condition of apoptosis, and the expressions of mRNA of bcl-2 and bax genes were assayed with RT-PCR. It was found that the apoptosis of human U937 cells could be induced by E.coli at various concentration ratios between cells and bacteria for 30 min in a dose-dependent manner. The apoptotic rates at cell/bacteria ratios of 0, 1∶5, 1∶10, 1∶20, 1∶50 and 1∶100 were 3.16%±0.90%, 9.46%±0.84%, 17.90%±1.41%, 35.59%±3.76%, 38.35%±7.12% and 55.07%±5.82% respectively. Also, there was a tendency of alterations in the expression levels of bcl-2 and bax genes with an increased expression level of bax gene and a reduced expression level of bcl-2 gene. It is concluded that E.coli can induce apoptosis in human U937 cells with a down-regulated expression of Bcl-2 and an up-regulated expression of Bax, and this might be related to the induction of apoptosis of the infected cell.

重组体pET-28a-alkB/E.coli降解页岩油泥石油烃的功能研究

为了降解页岩油泥中的石油烃,构建基因工程菌,表达石油烃降解关键酶以提高油泥的降解效果。以Pseudomonas qingdaonensis基因组为模板扩增alkB基因,连接至载体pET-28a,转化至E.coli BL21(DE3)中,SDS-PAGE和Western-blot鉴定表达的外源蛋白。pET-28a-alkB/E.coli处理原油和页岩油泥,采用气相色谱法评估降解效果。发现alkB基因在大肠杆菌中能表达外源蛋白,且14 d原油及页岩油泥的降解率分别为47.5%和47.1%,表明重组体pET-28a-alkB/E.coli具备降解页岩油泥石油烃的功能。

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.

Expression Pattern, Interaction Network, and Functional Analysis of the Arabidopsis Botrytis Susceptible1 Interactor

E3 ubiquitin ligases are participated in numerous processes, regulating the response to biotic and abiotic stresses. Botrytis susceptible1 interactor (BOI) is a RING (Really Interesting New Gene)-type E3 ligase that mediates the ubiquitination of BOS1 (Botrytis susceptible1), a transcription factor involved in stress and pathogen responses. Although BOI is an E3 ligase, there are reports to show that BOI interacts with target proteins such as DELLAs or CONSTANS to repress gibberellin responses and flowering without the degradation of the target proteins. In this article, we utilize diversified methods to comprehensively analyze the expression pattern, interaction network and function of BOI gene. Firstly, 1800 bp upstream region of BOI gene from Arabidopsis thaliana (Arabidopsis) genome was isolated, and fused GUS reporter gene. The resulting expression cassette was introduced into wild-type Arabidopsis through Agrobacterium-mediated transformation. The result demonstrated that BOI gene was expressed predominantly in leaves, siliques, young roots, and flowering tissues, indicating that BOI gene may be involved in multiple processes in plant growth and development in Arabidopsis. Besides, eight candidate interacting proteins were obtained from the Arabidopsis cDNA library via yeast two-hybrid technology, including EXO70E2 (AT5G61010), WRKY7 (AT4G24240), WRKY11 (AT4G31550), WRKY17 (AT2G24570), UBP20 (AT4G17895), L5 (AT1G12290), SAUR9 (AT4G36110) and TCP21 (AT5G08330). Functional analysis of these candidate interacting proteins manifested that they related to multiple pathways, including biological and abiotic stress, programmed cell death, protein degradation, material metabolism and transcriptional regulation. In addition, the results of the transient assay proclaimed that BOI protein affects the protein stability of EXO70E2 and L5 through its E3 ubiquitin ligase activity. Our results provide novel clues for a better understanding of molecular mechanisms underlying BOI-mediated regulations.

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.

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.

养殖场空气中E.coli磺胺类抗生素的抗性

本文从养殖场空气中分离出360株E.coli(大肠杆菌,Escherichia coli),应用肉汤微量稀释法和PCR方法,分离磺胺甲唑敏感菌株,检测抗生素抗性和抗性基因.在分离的E.coli中,对磺胺甲唑敏感菌株为95株(26.4%),有48株含有青霉素、氯霉素、四环素、环丙沙星、庆大霉素和利福平的抗性,而47株未含有抗性.其中,7株菌株含有1种抗生素抗性、11株菌株含有2种抗生素抗性、17株菌株含有3种抗生素抗性、6株菌株含有4种抗生素抗性、4株菌株含有5种抗生素抗性、3株菌株含有6种抗生素抗性.对抗生素的耐受能力依次为:氯霉素、青霉素、四环素、环丙沙星、庆大霉素、利福平.磺胺甲唑敏感菌株共检出163个抗性基因,sul1、int1、sul2、Int2、sul3检出数量依次为49、44、29、20和19;含一种、二种、三种、四种、五种抗性基因菌株分别为45、22、10、7、2;但有6株未检测出抗性基因.结果表明养殖场建场时间、抗生素使用、养殖规模等与抗生素抗性菌的抗性呈正相关;养殖场空气中分离的E.coli抗生素抗性较高,且具有多重抗性;抗生素抗性的表现型与其基因型之间出现不完全吻合现象.

秦皇岛地区狐源致病性E.coli对四环素类药物耐药性和耐药基因的检测

为了确定秦皇岛地区狐源大肠杆菌(E.coli)对四环素类药物的耐药性和耐药基因分布,采用常规的鉴定方法,从不同养狐场送检的腹泻的狐狸体内分离鉴定出20株E.coli。致病性试验表明,该菌为致病性E.coli。药敏试验结果表明:分离菌株对四环素和强力霉素药率分别达到95%和90%。通过PCR方法检测分离菌株四环素类药物的耐药基因,结果显示,tet A和tet B基因的检出率分别为100%和95%。本研究为秦皇岛地区防治狐源致病性大肠杆菌病提供实验基础。

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