• IMMUNE NETWORK
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• v.3 no.1
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• pp.16-22
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• 2003

Background: The S100A2 gene, also known as S100L or CaN19, encodes a protein comprised of 99-amino acids, is a member of the calcium-binding proteins of EF-hand family. According to a recent study, this gene was over-expressed in several early and malignant carcinomas compared to normal tissues. To elucidate the role of S100A2 protein in the process during carcinogenesis, production of monoclonal antibody specific to the protein is essential. Methods: First, cDNA sequence coding for ORF region of human S100A2 gene was amplified and cloned into an expression vector to produce GST fusion protein. Recombinant S100A2 protein and subsequently, monoclonal antibody to the protein were produced. The specificity of anti-S100A2 monoclonal antibody was confirmed by immunoblot analysis of cross reactivity to other recombinant proteins of S100A family (GST-S100A1, GST-S100A4 and GST-S100A6). To confirm the relation of S100A2 to cervical carcinogenesis, S100A2 protein in early cervical carcinoma tissue was immunostained using the monoclonal antibody. Results: GST-S100A2 recombinant protein was purified by affinity chromatography and then fusion protein was cleaved and S100A2 protein was isolated. The monoclonal antibody (KK0723; Korean patent pending #2001-30294) to the protein was produced and the antibody did not react with other members of EF-hand family proteins such as S100A1, S100A4 and S100A6. Conclusion: These data suggest that anti-S100A2 monoclonal antibody produced in this study can be very useful for the early detection of cervical carcinoma and elucidation of mechanism during the early cervical carcinogenesis.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.274-281
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• 2006

A fibrinolytic enzyme gene was isolated from Bacillus subtilis BB-1 by PCR method. Primers for PCR cloning were designed according to pre-identified gene for fibrinolytic enzymes from B. subtilis. The primer sequences were 5'-CGG ATC CGT GAG AGG CAA AAA GGT G-3' and 5'-TGA ATT CTT AAT GTG CTG CTG CTT GTC C-3' as concensus sequences of the fibrinolytic genes of Bacillus species. The PCR product was 1,145 bp and the sequence homology was 99% with nattokinase gene isolated from Japanese natto. The cloned fibrinolytic gene was reconstructed in Bacillus-E. coli shuttle vector, pEB for bulk-production. The fibrinolytic enzyme was purified by FPLC from the cloned B. subtilis 168. The optimum pH and temperature of the enzyme were 7.0 and $35^{\circ}C$, respectively. The fibrinolytic enzyme did not show any activity toward to skim milk, gelatin, casein and blood agar plate. The enzyme specific polyclonal antibody was prepared in rabbit for further assays such as detection of the gene expression in plant cells. This means that the enzyme may be used for health-care such as thrombosis without any hamful effects in the blood vessel.

• Journal of Mushroom
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• v.20 no.4
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• pp.199-207
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• 2022

The demand for novel strains has been rising in the domestic market to increase the production of sclerotia from Wolfiporia hoelen. To improve strain breeding efficiency, we investigated whether single-nucleotide polymorphisms (SNPs) in the RNA polymerase II subunit (RPB2) gene, which may be linked to the mating type locus, are useful for distinguishing monokaryons from dikaryons in Korean W. hoelen strains. We designed a specific primer set to efficiently amplify a region of RPB2 using PCR with the genomic DNA of 12 cultivated strains and 31 wild strains of W. hoelen collected from Korea. Nucleotide sequences of the PCR-amplified RPB2 genes were determined and analyzed for the presence of SNPs among the 43 W. hoelen strains. Previously reported SNP loci were detected in the RPB2 gene of all W. hoelen strains tested. However, these previously reported SNP loci could not be applied to differentiate monokaryons from dikaryons in approximately one-third of Korean wild strains with homozygous genotypes. Three additional SNPs in the RPB2 gene, which may improve the ability to distinguish monokaryons from dikaryons, were identified by searching through the multiple sequence alignments of the 43 W. hoelen strains. The applicability of these three novel SNPs, together with the previously known SNPs, in the RPB2 gene to W. hoelen strain breeding was verified by examining the hybrid strains and their parental strains.