Document Details
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Document Type |
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Thesis |
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Document Title |
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Immunological and Immunochemical studies on Phospholipase A2 from two snake venoms of Egypt دراسات مناعية وكيمياء مناعية على إنزيم الفسفوليبييز أ2 في سموم نوعين من الثعابين المصرية |
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Subject |
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Immunochemistry |
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Document Language |
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English |
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Abstract |
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The PLA2s of Naja haje haje and Pseudocerastes persicus fieldi venoms were purified, identified and their antigenicity and immunogenicity were investigated. The P. fieldi venom contained at least five PLA2 isoenzymes of quite different pI values, while the N. haje contained tow or more isoenzymes of quite close pI values. The molecular weight of the PLA2 variants of the tow venoms in SDS-PAGE were close to 14 KDa, whereas the major PLA2s of the N. haje and P. fieldi were focused at 9.2, 8.4 pIs and 5.6, 6.9, 8, 8.8, 9.8 pIs respectively. The major hemolytic PLA2 variant of the tow venoms were separated in four successive steps, which include gel filtration on Sephadex G-50, ion exchange chromatography on CM-Sephadex-CMS, preparative isoelectric focusing and reverse phase HPLC-C4 column. In the first tow steps, the hemolytic PLA2s fractions, (the N. haje-CMS and P. fieldi-CMS) were separated from the N. haje and P. fieldi respectively. The PLA2 variants of the N. haje-CMS and P. fieldi-CMS fractions were purified to homogeneity by preparative isoelectric focusing and reverse-phase HPLC chromatography on C4 column respectively. The protonated molecular ions [M+H]+ were obtained at m/z 13396, for N.haje-PLA2II; 13720, for P. fieldi-PLA2I; 13722, for P. fieldi-PLA2II; 13723; for P. fieldi-PLA2III; and 13718, for P. fieldi-PLA2IV, respectively. The complete amino acid sequence of P. fieldi FI, P. fieldi FII PLA2s and the partial sequences of the N. haje FII, P. fieldi FIII, and P. fieldi FIV PLA2s were obtained. The N-terminal amino acid sequences of the P. fieldi- FI, FII, III and IV PLA2s are similar to that of the CbI alpha, the acidic subunit of the P. fieldi heterodimeric neurotoxin. The N-terminal amino acid sequence of the N. haje FII PLA2 is similar to the Naja melanoleuca PLA2. The value of the N. haje- CMS and P. fieldi-CMS as immunogens for raising therapeutic antisera and as antigens for in vitro evaluation of the antisera potency was evaluated. The ELISA antibody titers to the N. haje and the N. haje-CMS in ten therapeutic equine antisera revealed poor correlation with the lethality neutralization. However, the rabbit antibodies to the N. haje-CMS and P. fieldi- CMS, neutralized the lethality of the corresponding venoms, which demonstrates its value as immunogens rather than diagnostic antigens. The isolated N. haje-PLA2 was tested for its ability to cause pathological changes to myocardium, skeletal muscle and cardiac ganglia of albino mice. Electron microscopic study revealed myodegeneration in cardiac and skeletal muscles and reduction in synaptic vesicles population of preganglionic nerve terminals in cardiac ganglia. Ultra structural changes in tissues were dose dependent. The lower dose (4 mg/kg) of PLA2 produced mild myocardial changes, while higher dose (8 mg/kg) of PLA2 caused myocardial degeneration. The skeletal muscle lesions were more severe than the myocardial changes. Some of the myofibrils were severely disorganized and lack typical striated appearance, sarcomeres disrupted, most of mitochondria were vesiculated and destroyed. Intramuscular injection of N. haje-PLA2 induced elevation in IL-6 in muscles, but no elevation of TNF-α was detected. It is concluded that these cytokines do not have a significant role in the pathogenesis of the local pathological effects induced by N. haje venoms in mice, although this does not exclude the possibility that these cytokines play a role in other aspect of venom-induced local pathology, as well as in reparative and regenerative responses that takes place after the onset of tissue damage. |
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Supervisor |
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Dr. Ahmed Fikry Abdel Monem Wahby |
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Thesis Type |
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Doctorate Thesis |
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Publishing Year |
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1425 AH
2005 AD |
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Co-Supervisor |
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Dr. Abdel Hakim Saad |
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Added Date |
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Saturday, January 15, 2011 |
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Researchers
| عمرو السيد الحكيم | El Hakim, Amr El Sayed | Researcher | Master | aselhakim@hotmail.com |
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