Biological characteristics of Chinese hamster ovary cells transfected with bovine Prnp

A normal prion protein (PrPc) is converted to a proteaseresistant isoform by an apparent self-propagating activity in transmissible spongiform encephalopathy, a neurodegenerative disease. The cDNA encoding open reading frame (ORF) of the bovine prion protein gene (Prnp) was cloned from Korean cattle by PCR, and was transfected into Chinese hamster ovary (CHO-K1) cells using lipofectamine. The gene expression of the cloned cDNA was confirmed by RT-PCR and Western blotting with the monoclonal antibody, 6H4. Cellular changes in the transfected CHO-K1 cells were investigated using parameters such as MTT, lactate dehydrogenase (LDH), and superoxide dismutase (SOD) activities, as well as nitric oxide (NO) production, and an apoptosis assay. In the MTT and LDH assays, the bovine PrnP-transfectant showed a lower proliferation rate than the wild-type (p < 0.05). Production of NO, after LPS or ConA stimulation, was not detected in either transfectants or CHO-K1 cells. In SOD assay under ConA stimulation, the SOD activity of transfectants was 10 times higher than that of CHO-K1 cells at 6 h after treatment (p < 0.05). The genomic DNA of both the transfectants and control cells began to be fragmented at 6 h after treatment with cyclohexamide. Caspase-3 activity was reduced by transfection with the bovine Prnp (p < 0.05). Conclusively, the viability of transfectants expressing exogenous bovine Prnp was decreased while the capacities for cellular protection against antioxidative stress and apoptosis were increased.

Changes in patterns of antimicrobial susceptibility and class 1 integron carriage among Escherichia coli isolates

The worldwide use of antimicrobials in different fields has created enormous pressure for the selection of resistance among opportunistic bacterial pathogen. One hundred four E. coli isolates were collected and identified from swine with diarrhea in Korea during the period of 2002. The isolates showed highly resistant to streptomycin (99. 0%), tetracycline (97. 1%), neomycin (91. 3%)and carbenicillin (84. 6%)in antimicrobial susceptibility test. Moreover, all of the isolates showed multiple antimicrobial resistant to more than 3, and 85%of them were resistant to more than 7 of total 14 antimicrobial agents. In comparison with isolates in 1998, resistance to antimicrobials was more frequent among the isolates in 2002. Presence of class 1 integrons was investigated through amplification of the gene with PCR, and could be classified 8 groups by pattern of 4 different amplicons. Class 1 integrons were observed in 67 strains (64. 2%)of E. coli from swine in Korea. One and 1. 6 kbp of amplicons were revealed to contain aadA1 and aadB-aadA1 gene cassettes respectively. Two kbp of amplicon had three different gene cassettes, dhfrXII-orfF-aadA2, and 3. 0 kbp of amplicon includes aadB-cmlA1 gene cassettes.

Development of a novel antigen capture-ELISA using IgY against porcine interleukin-6 and its application

Interleukin-6 (IL-6) is introduced as a marker of disease. At present, a variety of method may be used to quantify expression of this protein. Antigen capture-ELISA is a sensitive and accurate quantification method previously used with ovine, rat, and human IL-6 proteins. However, it has never been reported to quantify porcine IL-6 protein using capture ELISA. In this study, we generated and characterized a set of IgY and mono-specific polyclonal antibodies to recombinant porcine IL-6 (rpIL-6), and combining these with a sensitive and specific capture-ELISA for a diagnostic purpose. cDNA encoding the mature protein coding region of porcine IL-6 was cloned and expressed with pQE-30UA expression vector. rpIL-6 was then expressed and purified by using Ni-NTA resin. Protein mass of 24 kDa was found with SDS-PAGE and the identity of the protein was confirmed by Western-blot. Production of polyclonal antibodies against rpIL-6 was performed using the purified rpIL-6 in mice and hens. An antigen capture-ELISA was developed with the antibodies after their extraction. To compare the IL-6 level in the different sanitary state of farms, pig sera were randomly collected and concentration of IL-6 in the sera was measured with the antigen capture-ELISA. The capture-ELISA with the optimal concentration of antibodies, in this study, was able to detect about 10 ng/ml of rpIL-6. IL-6 levels determined with the capture-ELISA in pig sera showed positive correlation with the sanitary states of the farms. These results suggested that the developed antigen capture-ELISA could be a good tool for the screening of microbial infection in pig farms.