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Molecular structural differentiation Of Dendrobium species (Orchidaceae)
Abstracts / Journal of Biotechnology 136S (2008) S751–S759
VIII3-P-013 Production of recombinant antigens, polyclonal antibodies for development of an ELISA assay to detect Salmonella spp. in food Linh Thuoc Tran ∗ , Tri Nhan Nguyen University of Natural Sciences, Vietnam national University - Ho Chi Minh City, Viet Nam E-mail address: [email protected] (L.T. Tran). Salmonella is one of the most important food-borne pathogens. We have developed an ELISA assay being suitable for detection of Salmonella spp. in food, using polyclonal antibodies which can detect most Salmonella serotypes. To produce the antibodies, the recombinant H antigens of S. Enteritidis (H:g,m) and S. Typhimurium (H:i), were utilized to inject into rabbit, due to the fact that they are immunodominant antigenic surface structures of Salmonella and represent for two groups of the most common H antigen types, alpha cluster and G complex (McQuiston et al., 2004). The genes coding for antigens (fliCgm and fliCi ) were cloned into vector pGEX-5X-1 and successfully expressed in the strain E. coli BL21(DE3). An amount of 2.5 mg of each GST-fused protein (GSTH:g,m and GST-H:i) could be harvested. After injecting the proteins to rabbit and collecting blood, 100 ml of antiserum of each H antigen was obtained and was run through protein A column to purify polyclonal antibody. Polyclonal antibody was successfully conjugated to HRP by using a two-step glutaraldehyde procedure (Tijssen, 1985) and used to optimize the sandwich ELISA procedure. The optimized sandwich ELISA protocol was applied to test 60 Salmonella strains isolated from food samples and check the cross-reactivity with some other bacteria in food. The result showed that 99% of samples were positive of Salmonella presence (OD492 > 0.3), and only 1% was negative (OD492 < 0.3). Sandwich ELISA of some other bacteria in food, including E. coli, Shigella spp., Vibrio parahaemolyticus, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, resulted in very low values of OD492 (<0.2). References McQuiston, J.R., Parrenas, R., Ortiz-Rivera, M., Gheesling, L., Brenner, F., Fields, P.I., 2004. Sequencing and comparative analysis of flagellin genes fliC, fljB, and flpA from Salmonella. J. Clin. Microbiol. 42, 1923–1932. Tijssen, P., 1985. Practice and theory of enzyme immunoassays. Laboratory Techniques in Biochemistry and Molecular Biology 4th., 15, pp. 242–246.
doi:10.1016/j.jbiotec.2008.07.1684 VIII3-P-014 Molecular structural differentiation Of Dendrobium species (Orchidaceae) Sz-Jie Wu 1,∗ , Yu-Shan Liu 2 , Yuan-Tay Shyu 1 1
Department of Horticulture, National Taiwan University, Taipei 10617, Taiwan 2 Industrial Technology Research Institute, Hsinchu 310, Taiwan E-mail address: [email protected] (S.-J. Wu).
Dendrobium sp. (Shi-Hu, Dendrobium) are commonly used as traditional herbal ingredients in Asian countries (Sze et al., 2008). Medicinal and ornamental used Dendrobium usually have similar appearance and brings difficulty in differentiation. In this study, both methods of molecular and scanning electron microscopy structural analysis were developed to identify and to differentiate them. The genetic relationship of eight Dendrobium species was studied based on sequence analysis of the trnL intron/ trnL-
S757
trnF IGS region of cpDNA and the ITS region of rDNA (Tsai et al., 2004; Xu et al., 2006). The DNA sequences of trnL intron/ trnLtrnF IGS region obtained from 8 accessions were aligned with other Dendrobium species and two outgroups, and five clusters were generated from the phylogenetic analysis. D. huoshanense was grouped together and D. moniliforme and D. kingianum (AF519958.1), which are normally used as traditional herbal ingredients. Six clusters were generated from the phylogenetic analysis based on the DNA sequences of ITS region. The leaf and stem anatomy of eight Dendrobium species was also observed by SEM. The granular mucilage and acicular grain among the vascular bundle were observed on D. huoshanense, D. moniliforme and other medicinal use Dendrobium species, but not on D. crumenatum. In this study, molecular sequences analysis armed with SEM observation of leaf and stem structural anatomy showed an easy possible way to differentiate medicinal species from those of ornamental species. References Sze, C.W., Zhang, Y.B., Shaw, P.C., But, P.H., Ng, T.B., Tong, Y., 2008. A DNA microarray for differentiation of the Chinese medicinal herb Dendrobium officinale (Fengdou Shihu) by its 5 S ribosomal DNA intergenic spacer region. Biotechnol. Appl. Biochem. 48, 149–154. Tsai, C.C., Peng, C.I., Huang, S.C., Huang, P.L., Chou, C.H., 2004. Determination of the genetic relationship of Dendrobium species (Orchidaceae) in Taiwan based on the sequence of the internal transcribed spacer of ribosomal DNA. Sci. Horticult. 101, 315–325. Xu, H., Wang, Z., Ding, X., Zhou, K.X., 2006. Differentiation of Dendrobium species used as “Huangcao Shihu” by rDNA ITS sequence analysis. Planta Med. 72 (1), 89–92.
doi:10.1016/j.jbiotec.2008.07.1685 VIII3-P-015 Quantitative analysis of valiolamine through derivatization with phenylisocyanate by high-performance liquid chromatography He Li 1 , Jae Ran Lee 1 , Chang-Joon Kim 1,∗ , Yong Keun Chang 2 , Sung Bae Kim 1 , Yang Gon Seo 1 1 Department of Chemical & Biological Engineering, GyeongSang National University, Jinju, Republic of Korea 2 Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea
E-mail address: cj [email protected] (C.-J. Kim). Valiolamine is an important chemical intermediate for the synthesis of voglibose, antidiabetic medicine, starting from validamycin A and has been also isolated from the fermentation broth of Streptomyces hygroscopicus subsp. (Floss et al., 2000). Nowadays, intensive effort has been made to produce valiolamine by the fermentation of Streptomyces cells using metabolic engineering tool. Therefore, quantitative analysis of valiolamine is crucial for quality control of voglibose production process or monitoring the fermentation process to produce valiolamine. Valiolamine has no significant UV absorption, or fluorescence. Derivatization by chromophoric reagents can increase the sensitivity of valiolamine detection. The commonly used derivatization reagents are 2,4-dinitrofluorobenzene (DNFB), o-phthalaldehyde (OPA), 2,4,6trinitrobenzene sulfonic acid (TNBS) or dansyl chloride (Chen et al., 2005; Kim et al., 2003). This study was aimed to develop a rapid, accurate and precise HPLC method for determination of valiolamine following pre-column derivatization with phenylisocyanate (PIC) in microbial culture medium. The reaction with phenylisocyanate was performed in the glass vial containing dried liquid sample
VIII3-P-013 Production of recombinant antigens, polyclonal antibodies for development of an ELISA assay to detect Salmonella spp. in food Linh Thuoc Tran ∗ , Tri Nhan Nguyen University of Natural Sciences, Vietnam national University - Ho Chi Minh City, Viet Nam E-mail address: [email protected] (L.T. Tran). Salmonella is one of the most important food-borne pathogens. We have developed an ELISA assay being suitable for detection of Salmonella spp. in food, using polyclonal antibodies which can detect most Salmonella serotypes. To produce the antibodies, the recombinant H antigens of S. Enteritidis (H:g,m) and S. Typhimurium (H:i), were utilized to inject into rabbit, due to the fact that they are immunodominant antigenic surface structures of Salmonella and represent for two groups of the most common H antigen types, alpha cluster and G complex (McQuiston et al., 2004). The genes coding for antigens (fliCgm and fliCi ) were cloned into vector pGEX-5X-1 and successfully expressed in the strain E. coli BL21(DE3). An amount of 2.5 mg of each GST-fused protein (GSTH:g,m and GST-H:i) could be harvested. After injecting the proteins to rabbit and collecting blood, 100 ml of antiserum of each H antigen was obtained and was run through protein A column to purify polyclonal antibody. Polyclonal antibody was successfully conjugated to HRP by using a two-step glutaraldehyde procedure (Tijssen, 1985) and used to optimize the sandwich ELISA procedure. The optimized sandwich ELISA protocol was applied to test 60 Salmonella strains isolated from food samples and check the cross-reactivity with some other bacteria in food. The result showed that 99% of samples were positive of Salmonella presence (OD492 > 0.3), and only 1% was negative (OD492 < 0.3). Sandwich ELISA of some other bacteria in food, including E. coli, Shigella spp., Vibrio parahaemolyticus, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, resulted in very low values of OD492 (<0.2). References McQuiston, J.R., Parrenas, R., Ortiz-Rivera, M., Gheesling, L., Brenner, F., Fields, P.I., 2004. Sequencing and comparative analysis of flagellin genes fliC, fljB, and flpA from Salmonella. J. Clin. Microbiol. 42, 1923–1932. Tijssen, P., 1985. Practice and theory of enzyme immunoassays. Laboratory Techniques in Biochemistry and Molecular Biology 4th., 15, pp. 242–246.
doi:10.1016/j.jbiotec.2008.07.1684 VIII3-P-014 Molecular structural differentiation Of Dendrobium species (Orchidaceae) Sz-Jie Wu 1,∗ , Yu-Shan Liu 2 , Yuan-Tay Shyu 1 1
Department of Horticulture, National Taiwan University, Taipei 10617, Taiwan 2 Industrial Technology Research Institute, Hsinchu 310, Taiwan E-mail address: [email protected] (S.-J. Wu).
Dendrobium sp. (Shi-Hu, Dendrobium) are commonly used as traditional herbal ingredients in Asian countries (Sze et al., 2008). Medicinal and ornamental used Dendrobium usually have similar appearance and brings difficulty in differentiation. In this study, both methods of molecular and scanning electron microscopy structural analysis were developed to identify and to differentiate them. The genetic relationship of eight Dendrobium species was studied based on sequence analysis of the trnL intron/ trnL-
S757
trnF IGS region of cpDNA and the ITS region of rDNA (Tsai et al., 2004; Xu et al., 2006). The DNA sequences of trnL intron/ trnLtrnF IGS region obtained from 8 accessions were aligned with other Dendrobium species and two outgroups, and five clusters were generated from the phylogenetic analysis. D. huoshanense was grouped together and D. moniliforme and D. kingianum (AF519958.1), which are normally used as traditional herbal ingredients. Six clusters were generated from the phylogenetic analysis based on the DNA sequences of ITS region. The leaf and stem anatomy of eight Dendrobium species was also observed by SEM. The granular mucilage and acicular grain among the vascular bundle were observed on D. huoshanense, D. moniliforme and other medicinal use Dendrobium species, but not on D. crumenatum. In this study, molecular sequences analysis armed with SEM observation of leaf and stem structural anatomy showed an easy possible way to differentiate medicinal species from those of ornamental species. References Sze, C.W., Zhang, Y.B., Shaw, P.C., But, P.H., Ng, T.B., Tong, Y., 2008. A DNA microarray for differentiation of the Chinese medicinal herb Dendrobium officinale (Fengdou Shihu) by its 5 S ribosomal DNA intergenic spacer region. Biotechnol. Appl. Biochem. 48, 149–154. Tsai, C.C., Peng, C.I., Huang, S.C., Huang, P.L., Chou, C.H., 2004. Determination of the genetic relationship of Dendrobium species (Orchidaceae) in Taiwan based on the sequence of the internal transcribed spacer of ribosomal DNA. Sci. Horticult. 101, 315–325. Xu, H., Wang, Z., Ding, X., Zhou, K.X., 2006. Differentiation of Dendrobium species used as “Huangcao Shihu” by rDNA ITS sequence analysis. Planta Med. 72 (1), 89–92.
doi:10.1016/j.jbiotec.2008.07.1685 VIII3-P-015 Quantitative analysis of valiolamine through derivatization with phenylisocyanate by high-performance liquid chromatography He Li 1 , Jae Ran Lee 1 , Chang-Joon Kim 1,∗ , Yong Keun Chang 2 , Sung Bae Kim 1 , Yang Gon Seo 1 1 Department of Chemical & Biological Engineering, GyeongSang National University, Jinju, Republic of Korea 2 Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea
E-mail address: cj [email protected] (C.-J. Kim). Valiolamine is an important chemical intermediate for the synthesis of voglibose, antidiabetic medicine, starting from validamycin A and has been also isolated from the fermentation broth of Streptomyces hygroscopicus subsp. (Floss et al., 2000). Nowadays, intensive effort has been made to produce valiolamine by the fermentation of Streptomyces cells using metabolic engineering tool. Therefore, quantitative analysis of valiolamine is crucial for quality control of voglibose production process or monitoring the fermentation process to produce valiolamine. Valiolamine has no significant UV absorption, or fluorescence. Derivatization by chromophoric reagents can increase the sensitivity of valiolamine detection. The commonly used derivatization reagents are 2,4-dinitrofluorobenzene (DNFB), o-phthalaldehyde (OPA), 2,4,6trinitrobenzene sulfonic acid (TNBS) or dansyl chloride (Chen et al., 2005; Kim et al., 2003). This study was aimed to develop a rapid, accurate and precise HPLC method for determination of valiolamine following pre-column derivatization with phenylisocyanate (PIC) in microbial culture medium. The reaction with phenylisocyanate was performed in the glass vial containing dried liquid sample