Identification of alkA gene related to virulence of Shigella flexneri 2a by mutational analysis.

AIM: In vivo induced genes are thought to play an important role during infection of host. AlkA was identified as an in vivo-induced gene by in vivo expression technology (IVET), but its virulence in Shigella flexneri was not reported. The purpose of this study was to identify the role of alkA gene in the pathogenesis of S. flexneri. METHODS: PCR was used to amplify alkA gene of S. flexneri 2a and fragment 028pKm. The fragment was then transformed into 2457T05 strain, a S flexneri 2a strain containing Red recombination system, which was constructed with a recombinant suicide plasmid pXLkd46. By in vivo homologous recombination, alkA mutants were obtained and verified by PCR and sequencing. Intracellular survival assay and virulence assay were used to test the intracellular survival ability in HeLa cell model and the virulence in mice lung infection model respectively. RESULTS: Deletion mutant of S. flexneri 2a alkA was successfully constructed by gamma Red recombination system. The mutant exhibited significant survival defects and much significant virulence defects in mice infection assay. CONCLUSION: AlkA gene plays an important role in the infection of epithelial cells and is a virulent gene of Shigella spp.

Studies on the Expression of Recombinant Human MCAF/GM-CSF Fusion Protein and Its Biological Activities.

With PCR technology and methods of DNA recombination in vitro, the fusion protein of GM-CSF with MCAF, the facto that has a directing effect on cells to a target, was generated by the construction of recombinant plasmids pMG 01, pMG 02 and pMG 03 with different nucleotide composition between its SD sequence and the initiation codon ATG under the control of lambdaP(R) P(L) promoter of vector pBV 220. Although no stable secondary structure exist in the translation initiation regions of all the recombinant plasmid constructed, the expression levels of the products with DH5 alpha (pMG 02) and DH5alpha (pMG 03) are much higher than that with DH5alpha (pMG01) which hardly expressed the product. The assay of Western blot indicated that the expressed product reacted with MCAF and GM-CSF antibodies, respectively. The assay of biological activities showed that the expressed product had apparent monocyte chemoattractant activity and supported the growth of the human GM-CSF-dependent cell line TF1, suggesting that the biological functions of MCAF and GM-CSF are compatible

Screening of Cellulose Binding Motif (CBM) from Phage Peptides Library.

Bacteriophages capable of binding cellulose matrix were screened from a 15-mer phage peptides library. In the deduced amino acid sequences of the screened phages, a characteristic region containing a conserved aromatic residue [tyrosine (Y) or phenylalanine (F)] was found, which is similar to the normal cellulose binding domains found in fungal and bacterial cellulose catalysase. Dot-ELISA showed that the phage containing sequence as SWYL has higher affinity to cellulose fibre than other phages, such as those containing sequences as CWYGNC, CWYGEC and XSWYDXXSWFSX. Results indicated that SWYL maybe a good candidate for cellulose binding motif. This work laid basis for further study on cellulose binding motif.

Simultneous Expression of CS3 Colonization Factor Antigen and Cholera Toxin B Subunit in Salmonella typhi.

A host-plasmid balancing system was established based on asd gene in an avirulent strain of Salmonella typhi to express enterotoxigenic E.coli surface antigen CS3 and V.cholerae toxin subunit B(CTB). The plasmid can be stably maintained in the host and can express CS3 and CTB in the host cell without any antibiotic selection, although expression level and growth characteristics of the recombinant strain expressing either CS3 or CTB are superior to that of the recombinant strain which expresses both of the antigens. Antibo-dies against CS3 and CTB can be detected in sera of mice immunized with recombinant bacteria either orally or subcutaneously, and mice immunized subcutaneously can be protected from challenging with virulent strain of Salmonella typhi. This work may be helpful in constructing multivalent recombinant vaccines for prevention of bacterial diarrhea.

[Identification of a protein interacting with Shigella flexneri IpaC invasin by yeast two-hybrid system].

Two-hybrid system was applied to screen proteins interacting with IpaC in the host cell. By using two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKT-IpaC. A human HeLa cDNA library was screened to isolate protein factors that might interact with IpaC. Among the 2 x 10(6) clones screened, 22 positive clones were picked out. Sequence analysis revealed that two of them contained cDNA fragments from collagenase. Subsequently the domain of IpaC interacting with collagenase fragment was identified. These results suggest that IpaC might play a role in some biological processes where collagenase is involved.

[A novel approach to study pathogenesis of pathogens in vivo--signature-tagged mutagenesis].

Signature-tagged mutagenesis (STM) is a novel approach to study pathogenesis of pathogens and to screen virulence genes with high throughput in vivo,which is based on whole genome of pathogen in question. In recent years, more than ten species of microbial pathogens have been screened with this technology. There are also unknown virulence factors being identified with exception of known virulence genes identified in all these screens. This article reviews the principle, advantages and current limitations,the requirements, modifications of STM, and to date virulence genes identified by this technology.

[Construction of SW480 cell model identifying Shigella virulent genes].

Signature-tagged mutagenesis (STM) is a novel technology with high throughput screening ability to identify virulent genes of pathogen in vivo. An appropriate animal or cell line model is one of prerequisites by exploiting this technique. In order to apply STM to Shigella flexneri, RC426 was constructed as an attenuated mutant with chloramphenicol resistance and aroA and virG genes inactivated by homologous recombination; another attenuated strain T32 was used as an oral S. flexneri 2a vaccine due to a spontaneous deletion in three loci (ipaBCDA, invA and virG) on the virulence plasmid. The wild type strain 2457T had the invasion ability into host cells. The three strains, RC426, T32 and 2457T, were mixed together to invade colon cancer cell line SW480, and the distinct strains were recovered and counted from cell lysates of invaded SW480 in different time. The results showed that there were statistically significant differences between the amounts of two attenuated strains recovered and that of virulent strain within 12h invasion, indicating SW480 was a suitable cell model for applying STM to screen virulent genes of Shigella flexneri.

[Cloning and expression of spinach glycolate oxidase in Escherichia coli].

The cDNA coding spinach glycolate oxidase (GO) was amplified by RT-PCR using the total RNA of spinach leaves as the template, and was cloned into cloning vector pMD18-T. After the DNA sequence was determined, the go gene was subcloned into E. coli expression vector pBV220, pET-22b(+), pTIG-Trx and pThioHisC. SDS-PAGE analysis revealed that the recombinant E. coli BL21 (DE3) (pTIG-Trx-GO) and E. coli BL21 (DE3) (pET-22b(+)-GO) expressed the predicted 38 kD glycolate oxidase, and the enzyme activity was also detected.