[Analysis of ERG11 gene mutation in Candida albicans].

OBJECTIVE: To study the relationship between fluconazole (FCZ)-resistance of Candida albicans and the mutation of ERG11 gene encoding FCZ-targeted enzyme. METHOD: Three strains of FCZ-susceptible and 10 FCZ-resistent C. albicans were isolated from the urethra, vagina, oropharynx, respiratory tract, prostate secretion and blood samples. ERG11 gene was amplified by PCR using C.albicans genomic DNA extracts as the templates and the DNA sequences of the PCR products were determined and compared using BLAST and Clustal-W softwares. RESULTS: The comparison of ERG11 gene sequences identified mutations at 21 sites in 13 strains, including 17 same-sense and 4 missense mutations. Base substitutions at the sites of 348 bp and 383 bp resulting in D116E and K128T conversion may take place in both drug-resistant and drug-susceptible strains. The point mutation at the site of 1309 bp of FCZ-resistant strain may cause V437I change, and the base inversion at 1320 bp may give rise to A/C heterozygosity mutant of ERG11 gene, probably resulting in N440K conversion. CONCLUSION: FCZ-resistance of C. albicans may be associated with point mutations of V437I and N440K in ERG11 gene, but not with the point mutations of D166E and K128T.

Secreted expression of dengue virus type 2 full-length envelope glycoprotein in Pichia pastoris.

The full-length envelope glycoprotein gene of dengue virus type 2 was cloned using an RT-PCR method from the infected C6/36 cells and inserted into pPICZaB vector. The recombinant plasmid was integrated into Pichia pastoris by electroporation and the expressed product was identified by SDS-PAGE and Western blotting. High-level secreted expression was performed by determining the Mut(+) phenotype and screening multi-copy integrants in the recombinant yeast cells. A recombinant protein with a molecular size of approximately 69 kDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed supernatant was able to bind with mouse polyclonal antibody or E-specific monoclonal antibody of dengue-2 virus. Purified E-poly (His)-tagged fusion protein was obtained from the expressed product by passing through a metal-chelating affinity chromatographic (MCAC) column. The results of Western blotting and solid-phase ELISA using dengue virus antibodies indicated that the purified recombinant E glycoprotein retained its antigenicity. High-level production of the recombinant E protein up to 100 mg/l indicates that P. pastoris is an efficient expression system for dengue virus full-length envelope glycoprotein.

Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections.

A rapid and accurate method for detection for common pathogenic bacteria in foodborne infections was established by using oligonucleotide array technology. Nylon membrane was used as the array support. A mutation region of the 23S rRNA gene was selected as the discrimination target from 14 species (genera) of bacteria causing foodborne infections and two unrelated bacterial species. A pair of universal primers was designed for PCR amplification of the 23S rRNA gene. Twenty-one species (genera)-specific oligonucleotide detection probes were synthesized and spotted onto the nylon membranes. The 23S rRNA gene amplification products of 14 species of pathogenic bacteria were hybridized to the oligonucleotide array. Hybridization results were analyzed with digoxigenin-linked enzyme reaction. Results indicated that nine species of pathogenic bacteria (Escherichia coli, Campylobacter jejuni, Shigella dysenteriae, Vibrio cholerae, Vibrio parahaemolyticus, Proteus vulgaris, Bacillus cereus, Listeria monocytogenes and Clostridium botulinum) showed high sensitivity and specificity for the oligonucleotide array. Two other species (Salmonella enterica and Yersinia enterocolitica) gave weak cross-reaction with E. coli, but the reaction did not affect their detection. After redesigning the probes, positive hybridization results were obtained with Staphylococcus aureus, but not with Clostridium perfringens and Streptococcus pyogenes. The oligonucleotide array can also be applied to samples collected in clinical settings of foodborne infections. The superiority of oligonucleotide array over other tests lies on its rapidity, accuracy and efficiency in the diagnosis, treatment and control of foodborne infections.

[Fluconazole resistance in Candida albicans assayed by PCR fingerprinting with M13 prime].

OBJECTIVE: To understand the molecular and genetic mechanism underlying fluconazole resistance in Candida albicans by PCR fingerprinting with M13 primer. METHODS: Paper disc diffusion method was employed for assay of fluconazole resistance in 41 clinical isolates of Candida albicans, followed by PCR fingerprinting with M13 primer to study the gel patterns with cluster analysis using neighbor joining (NJ) method performed with RAPD200 software. RESULTS: Of the 41 clinical isolates, 11 strains (26.8%) were fluconazole-sensitive, 8 (19.5%) fluconazole-dependent and 22 (53.7%) fluconazole-resistance. Two to twelve bands could be observed among these strains, and the gel patterns revealed by cluster analysis were associated with the reactions of the strains against fluconazole and the location of infection. CONCLUSION: There is high prevalence of fluconazole resistance in clinical Candida albicans isolates, and PCR fingerprinting with M13 primer is convenient for assay of fluconazole resistance and molecular epidemiological study of Candida albicans.

Secreted expression and purification of dengue 2 virus full-length nonstructural glycoprotein NS1 in Pichia pastoris.

The dengue 2 virus (DEN-2) RNA (NGC strain) was used as a substrate to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA followed by polymerase chain reaction amplification of the NS1 region. Products were cloned into pPICZalphaB vector for sequencing and into Pichia pastoris for expression. A recombinant protein with a molecular size of approximately 80 KDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed protein was able to bind with mouse polyclonal antibody or NS1-specific monoclonal antibody of dengue 2 virus. Purified NS1-poly(His)-tagged fusion protein was obtained from the expressed product by passing through a metal-chelating affinity chromatographic (MCAC) column. The study also verified that our purified rNS1 protein retained its antigenicity. High-level production of the rNS1 protein up to 70 mg/l indicates that P. pastoris is an efficient expression system for dengue virus full-length NS1 glycoprotein.

Dengue virus type 2 infects human endothelial cells through binding of the viral envelope glycoprotein to cell surface polypeptides.

The endothelial cell line ECV304, derived from human umbilical cord and identified to be susceptible to dengue virus type 2 (DEN-2) infection, was used to study the molecular mechanism of DEN-2 binding to endothelial cells. DEN-2 was found by virus overlay protein-binding assays (VOPBAs) to bind to three ECV304 cell membrane proteins with molecular masses of 29, 34 and 43 kDa. Only a single protein of 29 kDa was observed when VOPBAs were carried out using preparations of trypsin-treated ECV304 cells. Pre-incubation of live ECV304 cells in culture or cell membrane proteins in modified VOPBAs with the recombinant DEN-2 envelope glycoprotein (rEgp) inhibited DEN-2 infection and blocked virus binding to the three proteins identified. These results indicate that DEN-2 rEgp could bind to three proteins on the surface of ECV304 cells. This virus-cell interaction may be associated with the receptor complex specific for DEN-2 infection of endothelial cells.