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5190 Afr. J. Microbiol. Res. 70 kda 60 kda 50 kda 1 2 3 4 5 6 7 Figure 1. Expression and purification of recombinant tcpB Ni-NTA purification of recombinant toxin co-regulated pilus produced in E. coli and stained with coomassie brilliant blue. Lane1: marker, lane 2: uninduced cells of pET32a-tcpB without using IPTG, lane 3, 4: induced cells of pET32a-tcpB with using IPTG at a long time 2 and 4 hours, lane 5, 6 and 7: Extract proteins after Ni-NTA affinity chromatography. incubation was continued for a further 4 h. The expressed protein was purified using Ni-NTA column according to manufacturer’s instruction (Qia gene). The purified protein was dialyzed twice against PBS (pH 7.5) at 4°C overnight. The quality and quantity of purified recombinant tcpB protein was dialyzed by SDS-PAGE (15%) and Bradford methods, respectively. Antigenicity and Immunoblot analysis of recombinant tcpB For preparation of primary antibody 100 µg of emulsion containing bacteria and complete freund´s Adjuvant (50 µg vibrio cholerae + 50 µg complete freund´s Adjuvant sigma, St. Louis Ma) at three weeks were injected into five mice, rabbit and acute phase patients and sera received as a gift from Dr. Amozande (Immunology Department, Iran, Arak). After 21 days Injections were repeated by replacing incomplete freund´s Adjuvant (50 µg vibrio cholerae + 50 µg incomplete freund´s Adjuvant). 10 days after the second injection, sera separated were used as primary antibodies. RESULTS DNA extraction The Chromosomal DNA of Vibrio cholera was extracted and concentration was adjusted to 250 µg/ml. This DNA was used as a template for amplification of tcpB gene. PCR product had expected size of 1297 bp compare to 100 bp DNA ladder (Fermentas). The sequencing result was confirmed by comparing with databases and using basic local alignment search tool (BLAST) soft ware (data not shown). Expression and purification of recombinant tcpB pET32a -tcpB in E. coli BL21 (DE3) plysS was induced and the expression protein was purified by Ni-NTA column (Figure 1). SDS.PAGE analyses, showed the expected molecular mass of near 65 kDa recombinant protein. The concentration of recombinant protein was Assayed and calculated to 400 mg purified protein per liter of the initial culture. Western immunoblot analysis To determine the antigenicity of recombinant tcpB in mice, human and rabbit immunized with Vibrio cholera, the recombinant tcpB was assayed by Western-blotting. Figure 2 shows the specific interaction between standardized antibody and purified recombinant tcpB
protein. DISCUSSION 70 kda 60 kda 50 kda Figure 2. Western blotting analysis of tcpB extracts using anti- tcpB rabbit, human and rabbit antiserums. lane 1 : Protein marker, lane 2 : interaction between serum of Immunized mice with purified recombinant tcpB protein , lane 3 : interaction between serum of Infected human with purified recombinant tcpB protein, lane 4: interaction between serum of Immunized rabbit with purified recombinant tcpB protein, lane 5 : control negative. Cholera is an acute diarrheal disease leading to death by severe dehydration without appropriate treatment, especially in developing countries. Vibrio cholerae is mainly a fecal-orally transmitted and humans are the only known natural host. Cholera has been endemic in southern Asia. Cholera has spread in seven pandemic since 1817. In 2008, the WHO reported 190,130 cholera cases worldwide, associated with 5143 deaths (98% in Africa), but cholera is globally under-reported and the true disease burden is estimated to be in the millions. In addition to endemic outbreaks, sporadic outbreaks can occur whenever sanitation and clean water provisions are lacking, such as occurred in Zimbabwe between 2008 to 2009. The ability of V. cholerae to persist in water will continue to confound our ability to eradicate cholera and thus cholera vaccines are needed. V. cholerae utilizes adhesion factors some of which may remain to be elucidated, but may include O1 LPS; GlcNAc-binding protein (GbpA); a protein (tcpF) secreted by the toxin coregulated pilus (tcp) biogenesis apparatus; outer kiaie et al. 5191 membrane protein OmpU and cholera toxin (CT), although this has only been implicated in an adult rabbit model. Tcp facilitates inter-bacterial interactions that are important for colonization. An effective cholera vaccine could prevent colonization by inducing the production of antibodies that directly neutralize the function of key colonization factors and/or facilitate phagocytosis and killing through bacterial opsinization (Bishop and Camilli, 2011). Tcp of V. cholera belongs to a subgroup of type-4 fimbriae and is expressed by both classical and ELT or strains of the O1 serotype, as well as O139 Bengal (Manning, 1997). In the present study, we sought Antigenic activity the tcpB, within the virulence factors from V. cholerae serotype inaba. Toxin-Coregulated Pilus B (tcpB) would be expected to be important element in pathogenesis in V. cholera. Tcpb is a predicated major pilin subunit protein with a molecular mass of 49.5 kDa and some similarity to tcpA in this pilus (Manning, 1997). In this study, recombinant protein TcpB was expressed in E. coli BL-21 (DE3) plysS bacteria through expression vector pET32a mediated transformation. The presence of 6xHis tag and T7 tag to the N or C terminal of recombinant peptide causes increase near 20 kDa. Therefore, molecular weight of tcpB-pET32a fusion
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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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Page 77 and 78: Novel Swine-Origin Influenza A H1N1
Page 79 and 80: that it produces lactic acid, bacte
Page 81 and 82: Figure 2. DMRT Graph, effect of var
Page 83 and 84: African Journal of Microbiology Res
Page 85 and 86: Bacillus colony Fig 1. Colony morph
Page 87 and 88: thuringiensis isolate S1 (Figure 6)
Page 91 and 92: Figure 1. The Kinetic of P. aerugin
Page 93 and 94: Figure 3. DNA-dosimeter determined
Page 95 and 96: Relative Fluorescence Unit Figure 4
Page 97 and 98: Conclusion The public health risk i
Page 99 and 100: general are members of the normal i
Page 101 and 102: Table 2. Distribution of Nosocomial
Page 103 and 104: and also to assess the influence of
Page 105 and 106: Table 1. List of decamers used in R
Page 107 and 108: Figure 2. RAPD profile of Fusarium
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: pilus (tcp) that is a subtle of pol
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
Page 143 and 144: Figure 1. Overview the Yongxing isl
Page 145 and 146: standard, XSLJ1, XSLJ2, XSLJ5, XSLJ
Page 147 and 148: Table 1. primer information: sequen
Page 149 and 150: conventional method. The reasons fo
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 and 160: Table 1. Primer oligonucleotide seq
Page 161 and 162: Figure 2. Nucleotide and putative a
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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62% were genotype D, A (14%), C (6%
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Swimming time (s) 1000 800 600 400
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Hepatic glycogen (mg/g) Hemoglobin
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Table 2. Sporulation index. Sign In
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Table 4. Conidial size of different
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Table 6. Sporulation of Alternaria
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Table 7. Reaction of different geno
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Table 2. Susceptibility of the clin
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Table 4. Aminoglycoside resistance
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Young, and the Councils on Clinical
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oleaginous microorganisms have been
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Table 2. Actual and predicted value
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Table 4. Analysis of variance (ANOV
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Huang et al. 5273 Figure 3. Respons
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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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