Stomatal density is important for crop yield. In this paper, we studied the epidermal pattern factors (EPFs) related to stomatal development. Prokaryotic expression vectors were constructed to obtain EPFs. Then the relationship between EPFs and hydrogen sulfide (H2S) was established. First, AtEPF1, AtEPF2 and AtEPFL9 were cloned and constructed to pET28a vectors. Then recombinant plasmids pET28a-AtEPF1, pET28a-AtEPF2 and pET28a-AtEPFL9 were digested and sequenced, showing successful construction. Finally, they were transformed into E. coli BL21(DE3) separately and induced to express by isopropyl β-D-galactoside (IPTG). The optimized expression conditions including IPTG concentration (0.5, 0.3 and 0.05 mmol/L), temperature (28 °C, 28 °C and 16 °C) and induction time (16 h, 16 h and 20 h) were obtained. The bands of purified proteins were about 18 kDa, 19 kDa and 14.5 kDa, respectively. In order to identify their function, the purified AtEPF2 and AtEPFL9 were presented to Arabidopsis thaliana seedlings. Interestingly, the H2S production rate decreased or increased compared with the control, showing significant differences. That is, EPFs affected the production of endogenous H2S in plants. These results provide a foundation for further study of the relationship between H2S and EPFs on stomatal development, but also a possible way to increase the yield or enhance the stress resistance.
Arabidopsis ; genetics ; metabolism ; Arabidopsis Proteins ; genetics ; isolation & purification ; metabolism ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Hydrogen Sulfide ; metabolism ; Plasmids ; genetics ; Seedlings ; metabolism

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.
Animals ; Antigens, Viral/genetics* ; Biological Assay ; Chickens/immunology* ; Escherichia coli/genetics* ; Infectious bursal disease virus/immunology* ; Poultry Diseases ; Vaccines, Synthetic/isolation & purification* ; Viral Structural Proteins/immunology* ; Viral Vaccines/immunology*

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.
Antibodies, Monoclonal ; chemistry ; isolation & purification ; metabolism ; Antibody Specificity ; DNA-Binding Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; Escherichia coli Proteins ; metabolism ; Humans ; Peptides ; Recombinant Fusion Proteins ; genetics ; metabolism

Pectobacterium carotovorum subsp. carotovorum is one of the world's top ten plant pathogens, mainly infecting cruciferous economic crops and ornamental flowers. In this study, an antibacterial gene cpxP (Gene ID: 29704421) was cloned from the genome of Pectobacterium carotovorum subsp. carotovorum, and constructed on the prokaryotic expression plasmid pET-15b, and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3), then stability and bacteriostatic experiments of the purified CpxP protein were performed. The final concentration of IPTG was 1 mmol/L, obtaining high-efficiency exogenous expression of the CpxP protein. There was no other protein after purification, and the destined protein exhibited good thermal stability and pH stability. The antibacterial test results showed that the inhibition rate of the CpxP protein on carrot slice was 44.89% while the inhibition rate on potato slice was 59.41%. To further explain its antibacterial mechanism, studying the spatial structure of this protein can provide new ideas for the control of soft rot and new protein pesticide targets.
Anti-Bacterial Agents ; pharmacology ; Bacteria ; drug effects ; Bacterial Proteins ; isolation & purification ; pharmacology ; Escherichia coli ; genetics ; Membrane Proteins ; isolation & purification ; pharmacology ; Pectobacterium carotovorum ; genetics ; metabolism ; Plasmids ; genetics

Emerging antibiotic resistance is a major global health threat. The analysis of nucleic acid sequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistance determinants to inform molecular diagnostics and drug development. We collected genetic data (11,087 newly-sequenced whole genomes) and culture-based resistance profiles (10,991 out of the 11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including 18 main species spanning a time period of 30 years. Species and drug specific resistance patterns were observed including increased resistance rates for Acinetobacter baumannii to carbapenems and for Escherichia coli to fluoroquinolones. Species-level pan-genomes were constructed to reflect the genetic repertoire of the respective species, including conserved essential genes and known resistance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed to infer gene-drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into GEAR-base, a resource available for academic research use free of charge at https://gear-base.com.
Acinetobacter baumannii ; genetics ; isolation & purification ; Bacteria ; genetics ; isolation & purification ; Cell Culture Techniques ; methods ; Drug Resistance, Microbial ; genetics ; Escherichia coli ; genetics ; isolation & purification ; Genome, Bacterial ; Genotype ; Humans ; Internet ; Microbial Sensitivity Tests ; Phenotype ; Whole Genome Sequencing

OBJECTIVE: To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel. METHODS: After sonication and centrifuge of strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected. RESULTS: The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h. CONCLUSIONS Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.
Escherichia coli ; genetics ; Glucosides ; chemistry ; Humans ; Protein Domains ; Protein Stability ; Pyrophosphatases ; chemistry ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; chemistry ; isolation & purification ; TRPM Cation Channels ; chemistry ; isolation & purification ; Thrombin ; metabolism

Enterotoxigenic Escherichia coli (ETEC) causes neonatal and post-weaning diarrhea in pigs. In order to determine the risk factors, rectal/fecal swabs and visceral organs obtained from pig farms in two regions of South Africa were analyzed microbiologically against risk variables. Seventy-two percent of young pigs were found to be positive for ETEC toxin genes; estB (38.9%), estB/STAP (25%), and estB/LT (13.9%) were dominant. Risk factors for ETEC-diarrhea in pigs include: leaving sick piglets in a pen with healthy piglets [odds ratio (OR) = 33.52; P < 0.0001]; water spillage in pen (OR = 42.87; P < 0.0001); hypothermic piglets (OR = 7.29; P < 0.0001); runt piglets in pen with healthy littermates (OR = 3.65; P < 0.0001); and prolonged use of antibiotics (OR = 3.05; P = 0.05).
Animals ; Animals, Newborn ; Diarrhea ; epidemiology ; microbiology ; Escherichia coli ; genetics ; isolation & purification ; Escherichia coli Infections ; epidemiology ; microbiology ; veterinary ; Genes, Bacterial ; Prevalence ; Rectum ; microbiology ; Risk Factors ; South Africa ; Swine ; Swine Diseases ; epidemiology ; microbiology ; Weaning

Objective: To analyze the effect of the identification and evaluation of Escherichia (E.) coli and Shigella, based on the upstream flanking sequences of CRISPR1. Methods: Both CRISPR and cas sequences were obtained through the BLAST with repeating sequences against the publicly complete genome in GenBank that related to E. coli and Shigella. Clustal X was used to perform multi-sequences alignment of the flanking sequences. PCR method was used to amplify the upstream flanking sequences of CRISPR1 in order to appraise the effect of identification and evaluation of upstream flanking sequences on E. coli and Shigella, which were based on the upstream flanking sequences of CRISPR1. Results: The results showed that 73.4% of the strains containing the I-E CRISPR/Cas that belonged to the phylogroups A, B1, D while 8.4% strains carried the I-F CRISPR/Cas. Another 17.2% of the strains owned CRISPR3-4 (non-CRISPR/Cas) only belonged to the phylogroups B2. All the Shigella strains carried I-E CRISPR/Cas. More than 99% of similarity the CRISPR1 upstream-flanking sequences was seen in E. coli (except B2) and Shigella and E. coli (B2). Both sensitivity and specificity were greater than 91% after PCR amplification in the region to identify the E.coli and Shigella. Conclusion: The upstream of CRISPR1 could achieve a preliminary identification effect on E.coli and Shigella.
Clustered Regularly Interspaced Short Palindromic Repeats/genetics* ; DNA, Bacterial/genetics* ; Escherichia coli/isolation & purification* ; Genotype ; Humans ; Molecular Sequence Data ; Sequence Analysis, DNA ; Shigella/isolation & purification*

OBJECTIVETo study the changes in gut microbiota and serum D-lactate level and their significance in children with recurrent pneumonia.

METHODSThe stool and blood samples were collected from 30 children with recurrent pneumonia (recurrent group), 30 children with acute pneumonia (acute group), and 15 children receiving surgical operation (surgery group). The 16S rRNA fluorescent quantitative polymerase chain reaction (FQ-PCR) was applied to determine the numbers of Bifidobacterium and Escherichia coli in stool samples, and the ratio between the logarithmic values of the numbers of Bifidobacterium and Escherichia coli (B/E value) was calculated. The serum D-lactate level was measured, and correlation analysis was performed.

RESULTSThe recurrent group had a significantly lower number of Bifidobacterium and a significantly lower B/E value than the acute group and the surgery group (P<0.05), as well as a significantly higher number of Escherichia coli than the surgery group (P<0.05). There was no significant difference in the number of Escherichia coli between the recurrent group and the acute group. The recurrent group had a significantly higher serum D-lactate level than the acute group and the surgery group (P<0.05). In the recurrent group, B/E value was negatively correlated with serum D-lactate level (r=-0.539, P<0.05).

CONCLUSIONSChildren with recurrent pneumonia may have biological and mechanical barrier damage in the intestinal mucosa.

Bacteria ; classification ; genetics ; isolation & purification ; Bifidobacterium ; genetics ; isolation & purification ; Child ; Child, Preschool ; Escherichia coli ; genetics ; isolation & purification ; Feces ; microbiology ; Female ; Gastrointestinal Microbiome ; Humans ; Lactates ; blood ; Pneumonia ; blood ; microbiology ; pathology ; Recurrence

The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis.
Animals ; Antigens, Bacterial/*immunology ; Brucella abortus/*enzymology/immunology ; Brucellosis/diagnosis/*veterinary ; Cattle ; Cattle Diseases/*diagnosis ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli/genetics ; Malate Dehydrogenase/*genetics/*immunology/isolation & purification ; Mice ; Recombinant Proteins/genetics/*immunology