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5224 Afr. J. Microbiol. Res. 2000 1000 750 500 200 100 Figure 1. Amplification of ghrelin gene by RT-PCR. M, DL2, 000 DNA marker; Lane 1, PCR product of ghrelin amplification. encoded 103 amino acids. The signal peptide region included 26 amino acids in length. (GeneBank accession number: HM567312). Homology of the ghrelin gene Figure 3 revealed the amino acid sequences of ghrelin of crucian carp and other fishes. The ORF region of ghrelin gene in crucian carp presented a high similarity with those of goldfish (99%), common carp (89.4%) and zebra fish (78.8%), as all four species derived from the Cyprinidae family. However, it only showed 53.8% similarity with channel fish. Therefore, it can be concluded that this ghrelin gene was rather conserved among different fish species. mRNA expression M The expression levels of the ghrelin gene were shown in Figure 4. High expression levels were detected in the intestine and liver, followed by mesonephron, head kidney and the spleen, and the skin, heart, muscle, gill and pituitary gland showed relatively weak expression levels. 1 Construction and identification of the recombinant plasmid The mature peptide gene fragment was amplified from the recombinant plasmid of pMD-ghrelin by PCR with the size of 248 bp and inserted into bacterial expression vector of pET-32a. As a result, the prokaryotic expression plasmid pGh-32 was obtained. pGh-32 was amplified and purified to recycle, digested with BamHIand XhoIenzymes, and tested by 1% (w/v) agarose gels electrophoresis. The flow chart of the vector construction was shown in Figure 5. Expression of the target recombinant protein The expressed products detected with 15% SDS-PAGE and a 27.0 KDa protein band could be seen after staining with Coomassie brilliant blue R250 (Figures 6 and 7). IPTG at concentrations of 0, 0.1, 0.2, 0.3, 0.5, 0.7 and 1.0 mmol/L could efficiently induce the expression of pGh-32. SDS-PAGE indicated that the optimal concentration of IPTG was 0.3 mmol/L. The ghrelin gene expressed as early as 1 h after IPTG induction, attaining peak levels around 3 h (Figure 7), the ghrelin fusion protein was mainly soluble protein and appeared in the precipitate only in a small amount (Figure 8). DISCUSSION Ghrelin plays an important role in appetite, adjusting of energy metabolism and immune system. More recently, it has been reported to be related to human diseases (Vila et al., 2007). The cDNA cloning and sequence analysis as well as appraisal of all amino acid of the ghrelin have been reported in non-mammalian vertebrates. However, no information is available on the role of ghrelin in teleost diseases. In this study, we obtained 490 bp of ghrelin which encoded 103 amino acids of ORF from the intestine of crucian carp, and the ghrelin involved 26 amino acids of the signal peptide region. The mature peptide started immediately after the signal peptide and this indicated that protein will be secreted out of the cell after its synthesis (Von Heijne, 1992) which demonstrated it was the secreted protein. The the high similarity between crucian carp and other fishes was consistent with previous studies (Kaiya et al., 2003c). Many species like rainbow trout (Kaiya et al., 2003a), Janpanese eel (Kaiya et al., 2003b), and channel catfish (Kaiya et al., 2005) showed highest expression levels in the stomach. Because the crucian carp lacks a stomach, our result showed the highest expression of ghrelin mRNA was found in intestine which had been proved in Cyprinidae family (Unniappan et al., 2002). This finding indicated that ghrelin could play important roles in gastrointestinal hormone in the crucian carp.
Figure 2. Nucleotide and putative amino acids sequences of crucian carp ghrelin. Beneath the nucleotide sequence is the puttied amino acids sequence. The signal peptide region is the black box. Figure 3. Multiple alignment of the deduced amino acid sequences of ghrelin in crucian carp and other vertebrates. The multiple alignments were produced using ClustalX. ‘*’ indicates positions that have a single, fully conserved residue. ‘:’ and ‘.’ indicate positions that have strong and weak similarity, respectively. Amino acid identities (%) between crucian carp and other vertebrates are show at end of each aligned sequence. Previous study (Itakura et al., 1977) reported that it was a milestone in genetic engineering to make a foreign gene successfully expressed in E. coil with the advantage of rapid growth rate, capacity for continuous fermentation, and relatively low cost. The purpose of the current study was to obtain the high expression levels of target gene to facilitate further functional analysis. The 26-amino acid signal peptide was identified by SingalP v3.0 software, which was often useless but influenced the protein expression in prokaryotic system; therefore, we cloned a truncated gene encoding the target protein without signal peptide into prokaryotic vector pET-32a. SDS-PAGE performed in this study confirmed that a 27 KDa protein band could be seen after staining which indicated that the recombinant prokaryotic expression system of pGh-32 was constructed successfully. The product of crucian carp ghrelin gene was mainly Zhou et al. 5225 soluble protein, which was consistent with the pig ghrelin protein (Yang et al., 2005). Intriguingly, the ghrelin gene in Black-feather chicken was expressed in E. coli 2566 by pTYB11 prokaryotic expression plasmids and the ghrelin protein was cytorrhyctes (Dai et al., 2008). These could be explained that the vector, form or condition might impact the existing form of the ghrelin protein in the host cell, and further study needed to be continued. The output of ghrelin was relatively high (approximately 33% of the total bacterial proteins) and this was beneficial to industrial production. Conclusions In conclusion, the ghrelin gene was obtained by molecular cloning techniques and was successfully
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African Journal of Microbiology Res
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Editors Prof. Dr. Stefan Schmidt Ap
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Electronic submission of manuscript
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Fees and Charges: Authors are requi
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nces Table of Contents: Volume 6 Nu
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Table of Contents: Volume 6 Number
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Table 1. Overview of the Soil prope
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exactly. MICROBIAL BIOMASS IN ORGAN
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Table 3. Advantages and disadvantag
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analysis of polymerase chain reacti
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Enzyme assay Triplicate samples of
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Figure 2. Effect of a) incubation t
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Table 2. Plasmid profile. Emerenini
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Table 1. Properties and identity of
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more understandable by the fact tha
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fertilizers considered the most imp
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Table 3. Effect of NPK levels and s
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Table 4. Effect of NPK levels and s
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Table 5. Effect of NPK levels and f
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with mineral NPK on wheat plant. Eg
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to synthetic antibiotics. In spite
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Among these, the K. pneumonia and V
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Figure 5. Pseudomonas aeruginosa An
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pathogens of their environment (She
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cultivable indigenous fishes of Ind
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Table 1. Oxidative stress parameter
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of tularemia. Ann N Y Acad. Sci., 1
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al., 1998; Heubuelt 1929) have alre
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Table 2. Influence of organic compo
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NO2 NO2 Zare et al. 5131 Figure 4.
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Sundermeyer-Klinger H, Meyer W, War
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Table 1. Composition of Mueller-Hin
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dose level, and is effective antibi
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wearing a mask is useful during the
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Novel Swine-Origin Influenza A H1N1
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that it produces lactic acid, bacte
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Figure 2. DMRT Graph, effect of var
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Bacillus colony Fig 1. Colony morph
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thuringiensis isolate S1 (Figure 6)
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Figure 1. The Kinetic of P. aerugin
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Figure 3. DNA-dosimeter determined
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Relative Fluorescence Unit Figure 4
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Conclusion The public health risk i
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general are members of the normal i
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Table 2. Distribution of Nosocomial
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and also to assess the influence of
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Table 1. List of decamers used in R
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Figure 2. RAPD profile of Fusarium
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Page 111 and 112: Table 1. Biosurfactant production p
Page 113 and 114: Figure 3. The correlation of reduct
Page 117 and 118: To estimate the water content, stra
Page 119 and 120: Kraiem et al. 5183 Table 2. Effect
Page 121 and 122: log CFU/g log CFU/g log CFU/g 8.5 7
Page 123 and 124: delays to cooling and wrapping on s
Page 125 and 126: pilus (tcp) that is a subtle of pol
Page 127 and 128: protein. DISCUSSION 70 kda 60 kda 5
Page 131 and 132: Percentage repellency (%) Percentag
Page 133 and 134: Fumigant toxicity of essential oils
Page 135 and 136: Table 1. Primers used for PCR and s
Page 137 and 138: Table 3. Amino acid substitutions i
Page 139 and 140: composition in qnrB alleles. Althou
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Page 147 and 148: Table 1. primer information: sequen
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Page 153 and 154: Figure 1. Comparison of amplificati
Page 155 and 156: Table 3. Fruiting-body formation an
Page 157 and 158: Yokoyama E, Yamagishi K, Hara A (20
Page 159: Table 1. Primer oligonucleotide seq
Page 163 and 164: Figure 6. Ghrelin expression induce
Page 167 and 168: Table 1. The SNPs related to the pr
Page 169 and 170: Figure 2. The phylogenetic trees of
Page 171 and 172: a c Rex DNA Tang et al. 5235 Figure
Page 175 and 176: Table 1. Gender and division wise d
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Page 181 and 182: Swimming time (s) 1000 800 600 400
Page 183 and 184: Hepatic glycogen (mg/g) Hemoglobin
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Page 191 and 192: Table 6. Sporulation of Alternaria
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Page 199 and 200: Table 4. Aminoglycoside resistance
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Page 205 and 206: Table 2. Actual and predicted value
Page 207 and 208: Table 4. Analysis of variance (ANOV
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the Central Universities (Grant No.
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Figure 1. Mechanism of enzymatic co
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Enzymatic activity (U/mL) Enzymatic
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prunin so far. By using the HPLC me
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Figure 7. Enzymatic conversion of n
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vaccine, HBs-Ab is negative in all
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