April - June 2007 - Kasetsart University

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340 (B) (A) <strong>Kasetsart</strong> J. (Nat. Sci.) 41(2) Figure 2 The 450 base pairs of IL-2 cDNA sequence of a BALB/c mouse (A). Alignment of the predicted protein sequences of BALB/c mouse (DQ836354), C3HeB/FeJ mouse (AY147902.1), RF mouse (MMU41494), C57BL6/J mouse (MMU41504), CZECHII/Ei mouse (MMU41505), and BKL mouse (MMU41506) which were analyzed by GeneDoc (B). The homology of amino acid was marked by black stripes.
Expression of rmIL-2 and purification The recombinant plasmid containing pQE30/mouse IL-2 cDNA was induced with IPTG to produce rmIL-2. The bacterial extracts containing the rmIL-2 gene could be obtained according to the protein pattern from SDS-PAGE. The expressed protein tended to aggregate with the cell debris as observed in the insoluble fraction by SDS-PAGE. However, under denaturing condition during the protein extraction, the rmIL- 2 could be resolved and then purified by Ni-NTA column chromatography. The rmIL-2 obtained from Ni-NTA resin column was highly purified (Figure 3). The bands of fusion proteins on SDS- PAGE showed a molecular weigh of 19,000 Da and 38,000 Da for monomeric and dimeric forms, respectively. In addition, these two bands were positively reacted with the IL-2 specific monoclonal antibody by Western blotting (Figure 4). Cell proliferation assay The purified rmIL-2 was analyzed for <strong>Kasetsart</strong> J. (Nat. Sci.) 41(2) 341 their biological activities by measuring the XTT colorimetric assay. The splenocytes were stimulated with serial dilutions of the purified rmIL-2 compared with non-stimulated cell culture as a negative control. The result showed the increasing values of OD450 nm and proliferative response after adding rmIL-2 at the concentration range of 5-2,560 ng/ml. When 40 ng/ml of the rmIL-2 was added, the increasing rate was distinctively seen and reached the plateau at the concentrations of 640-2,560 ng/ml (Figure 5). Receptor binding assay Mouse splenocytes were used in this study to determine the ligand-receptor binding activity. Cell surface membrane was stained red with sulforhodamine B (SRB) while the rmIL-2 bound to the cell receptor showed fluorescence green of the FITC conjugate. No or less signal was observed in non-stimulated cells. However, the splenocytes stimulated by mitogen exhibited increasing signal with high expression of IL-2 receptors on the cell surface (Figure 6). Figure 3 Ni-NTA purification of the recombinant mouse IL-2, analyzed by SDS-PAGE, demonstrated the two bands 19 kDa monomeric and 38 kDa dimeric forms in the eluate fractions (E1-E3) compared with non-induced transformant (C/-), IPTG-induced transformant (C/+), washing fractions (W1-W3) and molecular weight markers (M).
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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
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1989). In monogastrics, the inclusi
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of all LLS samples in this study we
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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 and 134: fluorescent lamp at 1.5 kLux develo
Page 137 and 138: Wisconsin, USA). Total extracted RN
Page 139: RESULTS Cloning and sequencing of m
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
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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
Page 185 and 186: c ≤ c2 jq2 j n q p k p * 2, j , ,
Page 187 and 188: algorithm would consider RM 5, prio
Page 189 and 190: Kasetsart J. (Nat. Sci.) 41(2) 389
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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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April - June 2007 - Kasetsart University

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