Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.
A new approach to using experimental phages for typing methicillin-resistant S. aureus (MRSA) non-sensitive to the phages of International Basic Set (IBS) is described. The collection Includes phage 85, modified on a culture of MRSA, and 5 phages induced from MRSA strains isolated in clinics of Moscow in 1975-76. Firstly, the modified phage selects cultures according to the specific character of its restriction-modification system, then the induced phages differentiate the selected strains into 5 groups (1, 2, 3, 4, 5) based on the specificity of the prophages they contain. Group 1 strains can further be differentiated into 5 subgroups (A, B, C, D, E) by additional phages. Forty-one MRSA strains isolated in 1987-90 in various hospitals of Moscow showing no sensitivity to IBS phages, were lysed by the modified phage, 15 of them belonging to Group 2 and isolated in the traumatological hospital, 26 belonging to Group 1 and were circulating in the burn center. Twenty-three strains of Group 1 appertain to subgroup 1B and were isolated over a 4-year period from the burned surface of patients and from the throat of a medical staff carrier.
The lysogenicity of 49 strains of methicillin-resistant S. aureus (MRSA) isolated in Moscow clinics in the 1970s and '80s was studied by the method of mitomycin C induction. It was found that one strain had phage of serogroup B, 33 strains had serogroup F phages and 15 strains had phages of both serogroups. In the course of genetic crossing on nitrocellulose filters it was demonstrated that serogroups B and F prophages contained in recipient cells 1) increase the frequency of transfer of conjugative plasmid pG873 and 2) mobilize transfer of non-conjugative plasmids pE994 and rms7.
Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the selfassembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.
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