An about 700 bp DNA fragment was amplified from genome DNA of S. aureus TSTw by PCR. This fragment was cloned into pGEM-7Zf(+) and the recombinant plasmid was transformed into E. coli DH5 alpha. The sequencing result of the recombinant plasmid demonstrated that it contains seb gene with 717 bp (without signal encoding region of 81 bp) which has the same nucleotide sequence as described in literature. The seb gene was cloned into expression vector 7ZTS and was transformed into E. coli JM109 (DE3). The expression level of SEB was as high as 33.3% of the cell total proteins.
Cloning, Molecular ; Enterotoxins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; Genetic Engineering ; Recombinant Proteins ; biosynthesis

OBJECTIVE: To construct an eukaryotic expression vectors containing superantigen staphylococcal enterotoxin A (SEA) gene, and to identify its expression in laryngeal squamous carcinoma cells. METHOD: SEA full-length gene fragment was obtained from ATCC13565 genome of the staphylococcus, referencing standard strains producing SEA. Coding sequence of SEA was artificially synthetized. Than, SEA gene fragments was subcloned into eukaryotic expression vector pIRES-EGFP. The recombinant plasmid pSEA-IRES-EGFP was constructed and was transfected to laryngocarcinoma Hep-2 cells. Resistant clones were screened by G418. The expression of SEA in laryngocarcinoma cells was identified with ELISA and RT-PCR method. RESULT: The subclone of artificially synthetized SEA gene was subclone to eukaryotic expression vector pires-EGFP. Flanking sequence confirmed that SEA sequence was fully identical to the coding sequence of standard staphylococcus strains ATCC13565 in Genbank. After recombinant plasmid transfected to laryngocarcinoma cells, the resistant clones was obtained after screening for two weeks. The clones were selected. The specific gene fragment was obtained by RT-PCR amplification. ELISA assay confirmed that the content of SEA protein in supernatant fluid of cell culture had reached about Pg level. CONCLUSION The recombinant eukaryotic expression vector containing superantigen SEA gene is successfully constructed, and is capable of effective expression and continued secretion of SEA protein in laryngochrcinoma Hep-2 cells after recombinant plasmid transfected to laryngocarcinoma cells.
Cell Line, Tumor ; Enterotoxins ; genetics ; Gene Expression ; Genetic Vectors ; Humans ; Plasmids ; Superantigens ; genetics ; Transfection

Objective: The docking and superantigen activity sites of staphylococcal enterotoxin-like W (SElW) and T cell receptor (TCR) were predicted, and its SElW was cloned, expressed and purified. Methods: AlphaFold was used to predict the 3D structure of SElW protein monomers, and the protein models were evaluated with the help of the SAVES online server from ERRAT, Ramachandran plot, and Verify_3D. The ZDOCK server simulates the docking conformation of SElW and TCR, and the amino acid sequences of SElW and other serotype enterotoxins were aligned. The primers were designed to amplify selw, and the fragment was recombined into the pMD18-T vector and sequenced. Then recombinant plasmid pMD18-T was digested with BamHⅠand Hind Ⅲ. The target fragment was recombined into the expression plasmid pET-28a(+). After identification of the recombinant plasmid, the protein expression was induced by isopropyl-beta-D- thiogalactopyranoside. The SElW expressed in the supernatant was purified by affinity chromatography and quantified by the BCA method. Results: The predicted three-dimensional structure showed that the SElW protein was composed of two domains, the amino-terminal and the carboxy-terminal. The amino-terminal domain was composed of 3 α-helices and 6 β-sheets, and the carboxy-terminal domain included 2 α-helices and 7 antiparallel β-sheets composition. The overall quality factor score of the SElW protein model was 98.08, with 93.24% of the amino acids having a Verify_3D score ≥0.2 and no amino acids located in disallowed regions. The docking conformation with the highest score (1 521.328) was selected as the analysis object, and the 19 hydrogen bonds between the corresponding amino acid residues of SElW and TCR were analyzed by PyMOL. Combined with sequence alignment and the published data, this study predicted and found five important superantigen active sites, namely Y18, N19, W55, C88, and C98. The highly purified soluble recombinant protein SElW was obtained with cloning, expression, and protein purification. Conclusions: The study found five superantigen active sites in SElW protein that need special attention and successfully constructed and expressed the SElW protein, which laid the foundation for further exploration of the immune recognition mechanism of SElW.
Humans ; Enterotoxins/genetics* ; Superantigens/genetics* ; Catalytic Domain ; Selenoprotein W/metabolism* ; Receptors, Antigen, T-Cell

OBJECTIVES: To explore the molecular characteristics of Staphylococcus aureus (S. aureus) in children, and to compare the molecular characteristics of different types of strains (infection and colonization strains) so as to reveal pathogenic molecular markers of S. aureus. METHODS: A cross-sectional study design was used to conduct nasopharyngeal swab sampling from healthy children in the community and clinical samples from infected children in the hospital. Whole genome sequencing was used to detect antibiotic resistance genes and virulence genes. A random forest method to used to screen pathogenic markers. RESULTS: A total of 512 S. aureus strains were detected, including 272 infection strains and 240 colonization strains. For virulence genes, the carrying rates of enterotoxin genes (seb and sep), extracellular enzyme coding genes (splA, splB, splE and edinC), leukocytotoxin genes (lukD, lukE, lukF-PV and lukS-PV) and epidermal exfoliating genes (eta and etb) in infection strains were higher than those in colonization strains. But the carrying rates of enterotoxin genes (sec, sec3, seg, seh, sei, sel, sem, sen, seo and seu) were lower in infection strains than in colonization strains (P<0.05). For antibiotic resistance genes, the carrying rates of lnuA, lnuG, aadD, tetK and dfrG were significantly higher in infection strains than in colonization strains (P<0.05). The accuracy of cross-validation of the random forest model for screening pathogenic markers of S. aureus before and after screening was 69% and 68%, respectively, and the area under the curve was 0.75 and 0.70, respectively. The random forest model finally screened out 16 pathogenic markers (sem, etb, splE, sep, ser, mecA, lnuA, sea, blaZ, cat(pC233), blaTEm-1A, aph(3')-III, ermB, ermA, ant(9)-Ia and ant(6)-Ia). The top five variables in the variable importance ranking were sem (OR=0.40), etb (OR=3.95), splE (OR=1.68), sep (OR=3.97), and ser (OR=1.68). CONCLUSIONS The random forest model can screen out pathogenic markers of S. aureus and exhibits a superior predictive performance, providing genetic evidence for tracing highly pathogenic S. aureus and conducting precise targeted interventions.
Child ; Humans ; Staphylococcus aureus/genetics* ; Cross-Sectional Studies ; Enterotoxins/genetics* ; Staphylococcal Infections ; Whole Genome Sequencing

Bacillus cereus is a common foodborne pathogen. Accidently eating food contaminated by B. cereus will cause vomiting or diarrhea, and even death in severe cases. In the present study, a B. cereus strain was isolated from spoiled rice by streak culture. The pathogenicity and drug resistance of the isolated strain were analyzed by drug sensitivity test and PCR amplification of virulence-associated gene respectively. Cultures of the purified strain were injected intraperitoneally into mice to examine their effects on intestinal immunity-associated factors and gut microbial communities, to provide references for the pathogenic mechanism and medication guidance of these spoilage microorganisms. The results showed that the isolated B. cereus strain was sensitive to norfloxacin, nitrofurantoin, tetracycline, minocycline, ciprofloxacin, spectinomycin, clindamycin, erythrocin, clarithromycin, chloramphenicol, levofloxacin, and vancomycin, but resistant to bactrim, oxacillin and penicillin G. The strain carries seven virulence-associated genes including hblA, hblC, hblD, nheA, nheB, nheC and entFM, which are involved in diarrhea-causing toxins production. After infecting mice, the isolated B. cereus strain was found to cause diarrhea in mice, and the expression levels of immunoglobulins and inflammatory factors in the intestinal mucosae of the challenged mice were significantly up-regulated. Gut microbiome analysis showed that the composition of gut microbial community in mice changed after infection with B. cereus. The abundance of the uncultured_bacterium_f_Muribaculaceae in Bacteroidetes, which is a marker of body health, was significantly decreased. On the other hand, the abundance of uncultured_bacterium_f_Enterobacteriaceae, which is an opportunistic pathogen in Proteobacteria and a marker of dysbacteriosis, was significantly increased and was significantly positively correlated with the concentrations of IgM and IgG. These results showed that the pathogenic B. cereus carrying diarrhea type virulence-associated gene can activate the immune system by altering the composition of gut microbiota upon infection.
Animals ; Mice ; Bacillus cereus/metabolism* ; Food Microbiology ; Immunity, Mucosal ; Diarrhea ; Microbiota ; Enterotoxins/genetics*

OBJECTIVETo establish a triplex TaqMan real-time PCR system containing internal amplification control (IAC) to detect cholera toxin gene ctxA and enterotoxigenic Escherichia coli (ETEC)heat-labile enterotoxin gene elt.

METHODSPrimers and probes were designed based on the sequences of ctxA, elt and IAC. Both sensitivity and specificity were analyzed and interactions between different reactions were evaluated.

RESULTSThis system showed that the sensitivity of ctxA was 94 copies/reaction while the elt 79 copies/reaction and the amplification efficiency were 94.7% and 98.1%, respectively. Under the ratio of copy numbers on gene ctxA to elt as between 1 : 1-1 : 10, when both targets were detected, with impact was less on each other. However, when the amount of elt or ctxA was 100 times of IAC, the amplification of IAC was significantly inhibited.

CONCLUSIONThis system showed both satisfactory sensitivity and specificity, thus could be used to detect pathogenic bacteria in diarrhea stools. The detection of IAC could prompt the presence of PCR inhibitors in samples being tested.

Cholera Toxin ; genetics ; Enterotoxigenic Escherichia coli ; genetics ; Enterotoxins ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo detect the enterotoxin genes of Staphylococcus aureus (SA) isolated from clinical specimens and analyze the correlation between enterotoxin genes and drug resistance of SA.

METHODSThe mecA gene and enterotoxin genes A-F of clinical SA isolates were identified by polymerase chain reaction (PCR), and the genes were sequenced to investigate the correlation of these genes to drug resistance.

RESULTSThe detection rate of enterotoxin genes was 100% in 67 methicillin- resistant SA (MRSA), showing no significant difference from the rate in 57 methicillin-sensitive SA (MSSA) (83.5%, χ(2)=0.203, P>0.05). Of the 116 strains carrying enterotoxin genes (93.5%), the detection rates of SEA, SEB, SEC, SED and SEF were 90.5%, 6.9%, 61.3%, 5.2%, 25.9% and 93.5%, respectively, and none of the strains were positive for SEE gene. In these strains, 78 (67.2%) carried 2 or more enterotoxin genes, and the main genotypes were SEA and SEC (33.6%), SEA and SEF (7.8%), and SEA and SEC and SEF (13.8%). Compared with the strains carrying a single enterotoxin gene, those with multiple enterotoxin genes showed a higher drug resistance rate, among which 75% of the SA strains carrying SEA+SEC+SEF were resistant to SXT, significantly higher than the rates of SA carrying SEA (28.6%) and SEA+SEC (38.7%) (P<0.05). The SA strains carrying SEA+SEC+SEF and SEA+SEF showed significantly higher amikacin resistance rates than SA strain carrying SEA (75.0%, 77.0%, 21.5%, respectively, P<0.05).

CONCLUSIONClinical isolates of SA carrying multiple enterotoxin genes have a higher drug resistance rate than those with a single enterotoxin gene, suggesting the the important role of enterotoxin in multidrug resistance.

Drug Resistance, Multiple, Bacterial ; genetics ; Enterotoxins ; genetics ; Humans ; Staphylococcal Infections ; microbiology ; Staphylococcus aureus ; drug effects ; genetics ; isolation & purification

OBJECTIVETo investigate the limited digestion of recombinant staphylococcal enterotoxin C2 (SEC2-His)in different conditions.

METHODSThe purified recombinant SEC2-His was treated with different reagents and the cleavage of rSEC2 molecule was observed by SDS-PAGE.

RESULTThe cleavage occurred in positions Cys93-Cys110 of the disulfide loop. Complete auto-cleavage of recombinant SEC2 was observed in solution at 37degrees within 24 hrs, and that was accelerated under alkaline conditions. The auto-cleavage of the recombinant protein was inhibited in the presence of beta-ME (2%), PMSF (5-10 mmol/L), imidazole (1 mol/L) or crude E.coli lysate. Non-specific degradation of recombinant SEC2 was promoted with the increasing of the concentration of H(2)O(2).

CONCLUSIONThe recombinant SEC2-His is broken down in special site of protein, which may be associated with the protein structure.

Amino Acid Sequence ; Enterotoxins ; chemistry ; genetics ; Molecular Sequence Data ; Protein Conformation ; Protein Stability ; Recombinant Fusion Proteins ; chemistry ; genetics

OBJECTIVETo detect the "high pathogenicity island" of Yersinia enterocolitica WA in E. coli and the to provide evidence for theory base of bacterial evolution process and the different structures in different E. coil.

METHODSPolymerase chain reaction (PCR), nucleic acid hybridization in situ were used to detect and identify HPI. DNA sequencing was used to compare the gene homology of HPI among E. coli with Yersinia enterocolitica (Yen).

RESULTSThe irp2 and fyua genes of Yen HPI were investigated in E. coli strains. Among them, 30 strains were isolated from 93 Enterotoxigenic E. coli (ETEC) strains and 3 strains were positive in 10 strains Enteropathogenic (EPEC). HPI was also detected in Enteroaggregative E. coli (EAggEC) strain. In most of these isolates, HPI was bordered by an asntRNA locus, as in Yersinia sp. Through sequential comparison, the gene sequence homology was higher between in EPEC and EAggEC than ETEC and Yersinia enterocolica.

CONCLUSIONSETEC, EPEC and EAggEC were pathogenicity bacterias and many of them harboring HPI of Yen and the HPI had the same position in E. coli chromosome as Yersinia enterocolitica but the diversity of structure and sequence in these E. coli might suggest that the HPI of these different serotype E. coli were from different ancient bacterias. At the same time, the high positivity rate of HPI in E. coli might be crucial to virulence change, virulence evolution and virulence regulation in E. coli.

Bacterial Proteins ; analysis ; genetics ; Enterotoxins ; genetics ; Escherichia coli ; genetics ; pathogenicity ; Escherichia coli Infections ; microbiology ; Genes, Bacterial ; Receptors, Cell Surface ; genetics ; Virulence ; genetics ; Yersinia enterocolitica ; genetics ; pathogenicity

OBJECTIVETo enhance the expression level of staphylococcal enterotoxin O (SEO) by optimization of rare codons.

METHODSThe gene of mature SEO (His-tag included) was cloned to pET28a, and 15 rare codons on the gene were optimized by PCR technology. These recombinant plasmids then were transformed into E.coli BL21(DE3), respectively. After IPTG induced, the expression levels of those mutants were analyzed by SDS-PAGE. The proteins were purified and their bioactivities were determined.

RESULTAfter the optimization of rare codons, the expression levels were increased from 7.49% to 19.8% in total cell proteins. The optimized SEO had bioactivity to stimulate the proliferation of murine lymphocytes, which was equivalent to that of non-optimized SEO in vitro.

CONCLUSIONOptimization of rare codons can enhance the expression of SEO effectively.

Animals ; Cloning, Molecular ; Codon ; genetics ; Enterotoxins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Mice ; Mutation ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transformation, Bacterial