April - June 2007 - Kasetsart University

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334 Suzuki. 2001. Profile of polyunsaturated fatty acids production by Thraustochytrium sp. KK17-3. J. Am. Oil. Chem. Soc. 78: 605- 610. Iida, I., T. Nakahara., T. Yokochi., Y. Kamisaka., H. Yagi., M. Yamaoka. and O. Suzuki. 1996. Improvement of docosahexaenoic acid production in culture of Thraustochytrium aureum by medium optimization. J. Ferment. Bioeng. 81: 76-78. Johnson, E. A. and G.H. An. 1991. Astaxanthin from microbial sources. Crit. Rev. Biotechnol. 11: 297-326. Johnson, E.A. and M.J. Lewis. 1979. Astaxanthin formation by the yeast Phaffia Rhodozyma. J. Gen. Microbiol. 115: 173-183. Kinsella, J.E. 1987. Seafoods and Fish Oils in Human Health and Disease. New York: Marcel Decker. 320 p. Lewis, T.E., P.D. Nichols and T.A. McMeekin. 1999. The Biotechnological Potential of Thraustochytrids. Mar. Biotechnol. 1: 580- 587. Mo, C. and B. Rinkevich. 2001. A simple. Reliable and fast protocol for Thraustochytrid DNA extraction. Mar. Biotechnol. 3: 100-102. Marvelisa, L.C., T. Naganuma and Y. Yamaoka. 2003. Identification by HPLC-MS of carotenoids of Thraustochytrium CHN-1 strain isolated from Seto Inland Sea. Biosci. Biotechnol. Biochem. 67: 884-888 Ratledge, C. 2004. Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production. Biochimie. 86: 807-815. Schroeder, W.A. and E.A. Johnson. 1993. Antioxidant role of carotenoids carotenoids in Phaffia rhodozyma. J. Gen. Microbiol. 139: 907-912. Kasetsart J. (Nat. Sci.) 41(2) Simopoulos, A.P. 1989. Summary of NATO Advanced Research Workshop on dietary ω3 and ω6 fatty acids: biological effects and nutritional essentiality. J. Nutr. 119: 521-528. Takahata, K., K. Monobe, M. Tada and P.C. Weber. 1998. The benefits and risks of n-3polyunsaturated fatty acids. Biosci. Biotechnol. Biochem. 62: 2079-2085. Valadon, L.R.G. 1976. Carotenoids as additional taxonomic characters characters in fungi: A review. Trans. Br. Mycol. Soc. 67: 1-15. Vazques, M., V. Santos and J.C. Parajo. 1997. Effect of the carbon source on the carotenoid Profiles of Phaffia rhodozyma strains. J. Ind. Microbiol. and Biotech. 19: 263-268. White, T.J., T. Bruns., S. Lee., and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, pp. 315-322. In M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White (eds.). PCR Protocol : A Guide to Methods and Applications. Academic Press, San Diego. Wu, S.T., S.T. Yu, and L.P. Lin. 2005. Effect of culture conditions on docosahexaenoic acid production by Scizochytrium sp. S31. Proc. Biochem. 40: 3103-3108. Yamaoka, Y., M.L. Carmona and S. Oota. 2004. Growth and carotenoid production of Thraustochytrium sp. CHA-1 cultured under superbright red and blue light-emitting diodes. Biosci. Biotechnol Biochem. 68: 1594-1597. Yokochi, T., D. Honda., T. Higashihara. and T. Nakahara. 1998. Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR 21. Appl. Microbiol. Biotechnol. 49: 72-76. Yongmanitchai, W. and O.P. Ward. 1989. Omega- 3 fatty acids: Alternative sources of production. Proc. Biochem. 24: 117-125.
Kasetsart J. (Nat. Sci.) 41 : 335 - 345 (2007) Cloning, Expression, Purification and Biological Activities of Recombinant Mouse Interleukin-2 in E. coli M15 Sanchai Chantajorn 1 , Ratchanee Hongprayoon 2 * and Thaweesak Songserm 3 ABSTRACT Molecular cloning, sequencing and expression of recombinant mouse interleukin 2 (rmIL-2) were described. The interleukin-2 (IL-2) cDNA, 450 base pairs in length with repeating CAG, was amplified using specific primers. The IL-2 cDNA showed high homology at 100%, 100%, 91%, 96% and 94% with five strains of mice previously reported (GeneBank accession number AY147902.1, MMU41494, MMU41504, MMU41505 and MMU41506). The IL-2 gene was cloned into the pDrive cloning vector and consequently expressed using pQE30 expression vector which provided high expression level of the recombinant protein. The predicted rmIL-2 sequence is 161 amino acids with a molecular weight of 19 kDa. The expressed protein was then purified by Ni-NTA column under denaturing condition. Analysis of the rmIL-2 by SDS-PAGE demonstrated two bands of 19 and 38 kDa representing monomeric and dimeric forms of this protein. The biological activity in stimulating T-cell proliferation was also described and the binding signal to the receptor was easily observed by immunofluorescence. Key words: mouse interleukin-2, cloning, protein expression, protein purification, immunofluorescence INTRODUCTION Interleukin-2 (IL-2) is a growthpromoting activator for bone marrow-derived T lymphocytes (Smith, 1989), and was among the first cytokines to be characterized at the molecular level. Interleukin-2 was the major autocrine growth factor for T lymphocytes, and the quantity of IL-2 synthesized by activated CD4 + T cells was an important determinant of the magnitude of immune response. The action of IL-2 on T-cells was mediated by binding to IL-2 receptor proteins. This system was perhaps the best understood mechanism of all cytokine receptors (Morgan et al., 1976). Interleukin-2 exerts its effects on many cell types, the most prominent of which is the T lymphocyte. Indeed, one of the most rapid consequences of T cells activation through its antigen receptor is the de novo synthesis of IL-2. This was quickly followed by expression of a high affinity IL-2 receptor on surface membrane of CD4 + T cells, CD8 + T cells, B cells and natural killer (NK) cells (Lenardo et al., 1999). Interleukin-2 induce cells proliferation via pro- 1 Centre for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand. 2 Department of Plant Pathology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand. 3 Department of Veterinary Pathology, Faculty of Veterinary, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand. * Corresponding author, e-mail: agrrat@ku.ac.th Received date : 15/08/06 Accepted date : 13/02/07
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April - June 2007 Volume 41 Number
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KASETSART JOURNAL NATURAL SCIENCE T
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Kasetsart J. (Nat. Sci.) 41 : 205 -
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harvesting time which started from
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y Chen and Paull (2000). Figure 1 a
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pineapple fruit allowed the accumul
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Kasetsart J. (Nat. Sci.) 41(2) 215
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Cluster analysis Cluster analysis u
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Table 3 (Continued) 1 2 3 4 5 6 7 8
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CONCLUSION AFLP markers classified
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difference of soil texture used in
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under wet soil condition planting.
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tropics. Following the expansion, w
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sealed in a plastic box with 1 cm w
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moisture content increment after so
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Frequency Frequency 30 20 10 0 20.0
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School, Kasetsart University. The a
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for combining ability of lines (Lam
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selective mass sibbing within indiv
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Although most of S 2-interfamily hy
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composite sets as used in this stud
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days. The numbers of calli producin
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the report of Ching (1982) showing
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clearly amplified bands generated b
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caused by either the recombination
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Kuhlmann, V. and B. Foroughi-Wehr.
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Xanthomonas fuscans subsp. aurantif
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was collected and washed with 70% e
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expected size was amplified with 35
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eaction technique has been used for
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Schaad, N.W., D. Opgenorth and P. G
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MATERIALS AND METHODS Model descrip
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calculated TF value calculated TF v
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sincere gratitude and deep apprecia
Page 83 and 84: 1989). In monogastrics, the inclusi
Page 85 and 86: of all LLS samples in this study we
Page 87 and 88: Melbourne, Parkville, Victoria. Goe
Page 89 and 90: considered responsible for low prod
Page 91 and 92: Kasetsart J. (Nat. Sci.) 41(2) 291
Page 93 and 94: mineral supplementation suggested b
Page 95 and 96: Body weight chane (kg/head) 32 30 2
Page 97 and 98: CONCLUSION AND RECOMMENDATION With
Page 99 and 100: SAS. (Statistical Analysis System).
Page 101 and 102: genes covering a variety of desirab
Page 103 and 104: mg/l NAA and 0.5 mg/l zeatin) incub
Page 105 and 106: 5. Purification by various sucrose
Page 107 and 108: as the culture period increased. So
Page 109 and 110: culture, pp. 1-20. In L.C. Fowke an
Page 111 and 112: Kasetsart J. (Nat. Sci.) 41 : 311 -
Page 113 and 114: GC. Analytical methods Total carboh
Page 115 and 116: ash. The crude hot water polysaccha
Page 117 and 118: spirulan (H-SP), and a desulfated c
Page 121 and 122: cells often displayed an increased
Page 123 and 124: LITERATURE CITED Bell, T.A. and D.V
Page 125 and 126: for alternative sources of supply.
Page 127 and 128: BR2.1.2 formed the same clade with
Page 129 and 130: supplement with glutamate (Iida et
Page 131 and 132: optimal for S. limacinum BR2.1.2 fo
Page 133: fluorescent lamp at 1.5 kLux develo
Page 137 and 138: Wisconsin, USA). Total extracted RN
Page 139 and 140: RESULTS Cloning and sequencing of m
Page 141 and 142: Expression of rmIL-2 and purificati
Page 143 and 144: other strains of mice have differen
Page 145 and 146: dose interleukin-2 therapy promotes
Page 147 and 148: The chitosano-oligosaccharides are
Page 149 and 150: enzyme and cells were maximized by
Page 151 and 152: of crude chitosanases. Fortunately,
Page 153 and 154: Relative activity (%) 100 80 60 40
Page 155 and 156: Uchida, K. Tateishi, O. Shida and K
Page 157 and 158: MATERIALS AND METHODS 1. Materials
Page 159 and 160: without coating and coated with pec
Page 161 and 162: in Table 5-7. It was found that the
Page 165 and 166: the cultures at 10,200 × g for 15
Page 167 and 168: incubation and reached the final pH
Page 169 and 170: y lysine- and tyrosine-decarboxylas
Page 171 and 172: during the stages of ripening as su
Page 175 and 176: As the 25 companies were divided in
Page 177 and 178: All the data comes up with informat
Page 179 and 180: as piece “A” or straight as pie
Page 181 and 182: et al. (1998); Zeng (2000); and Tal
Page 183 and 184: p 1, i * Kasetsart J. (Nat. Sci.) 4
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c ≤ c2 jq2 j n q p k p * 2, j , ,
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algorithm would consider RM 5, prio
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the maximum deviation of about 10%.
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Stock Quantities Using Heuristic Op
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which dynamically and automatically
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2. Framework architecture and compo
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Risk (VaR) calculation which was im
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Throughput (job/sec) 7 6 5 4 3 2 1
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Speed up 40 35 30 25 20 15 10 5 0 F
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and discovery, resource selection a
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