ABSTRACT
OBJECTIVE To investigate the significance of CNS in clinical infections.METHODS A total of 114 CNS strains isolated from our hospital were identified by conventional procedures and the icaD gene was amplified by PCR.RESULTS Of all CNS strains,the highest isolated rate was S.epidermidis(41.2%).CNS isolated from deep venous catheters,wound secretions and blood had a higher rate of carrying ica operon,accounted for 44.4%,42.1% and 36.8%,respectively,whereas 24.0% in respiratory secretions and 14.1% in urine.Among the ica operon positive CNS strains,the percentages of S.epidermidis and S.haemolyticus were 42.6% and 19.0%.CONCLUSIONS There is a wide range of CNS species carrying ica operon,especially in S.epidermidis.CNS isolated from different specimens might have different significances.It should be cautious to assess the results of isolated CNS from respiratory and urine specimens.The CNS isolates from blood specimens might be contaminated.The PCR method for the ica operon is simple and easy.
ABSTRACT
Summary: A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E.coli DE3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28 % of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect ELISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.
ABSTRACT
To set up a method of amplification for the whole CagA gene of Helicobacter pylori and its fingerprinting by restriction fragment length polymorphism (RFLP), nested PCR was employed in combination with TD-PCR to amplify the gene and EcoRI and Hind III were used to generate the RFLP fingerprinting. Target DNA fragments from 13 of 20 samples were successfully amplified and the relevant RFLP fingerprintings were obtained. It is concluded that the method can be used to amplify the whole CagA gene and CagA gene has apparent diversity of RFLP profile.
Subject(s)
Humans , Antigens, Bacterial , Genetics , Bacterial Proteins , Genetics , DNA Fingerprinting , Methods , Gene Amplification , Genetics , Helicobacter pylori , Genetics , Polymorphism, Restriction Fragment LengthABSTRACT
To set up a method of amplification for the whole CagA gene of Helicobacter pylori and its fingerprinting by restriction fragment length polymorphism (RFLP), nested PCR was employed in combination with TD-PCR to amplify the gene and EcoRI and Hind III were used to generate the RFLP fingerprinting. Target DNA fragments from 13 of 20 samples were successfully amplified and the relevant RFLP fingerprintings were obtained. It is concluded that the method can be used to amplify the whole CagA gene and CagA gene has apparent diversity of RFLP profile.