Application Of New Technology For Preparation Of Fowl Cholera Vaccine

Transcription

1 Application Of New Technology For Preparation Of Fowl Cholera Vaccine

2 A thesis presented by

3 Under supervision of

4 Prof. Dr: Mohamed Kamal Refai Prof. of Microbiology Faculty of Vet. Med Cairo Univ.

5 Prof. Dr: Jaken K. A. El-Jakee Prof. of Microbiology Faculty of Vet. Med Cairo Univ.

6 Prof. Dr: ELHAM, A. EL-EBIARY Chief Researcher and Director of Central Laboratory For Evaluation of Vet. Biologics, Abbassia, Cairo.

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9 Fowl cholera vaccines Whole cell vaccines Living vaccines Killed vaccine Cell component vaccine

10 Outer membrane proteins vaccine OMP proteins OMP DNA vaccine

11 The current study aimed to: Studying the differences between the omph of P. multocida serotypes by PCR and RFLP Preparation of omph gene based DNA vaccine Evaluation of its vaccine potential

12 Studying the differences between the omph of P. multocida serotypes by PCR and RFLP

13 Extraction of genomic DNA of P. multocida strains Agarose gel electrophoresis PCR amplification of omph gene RFLP analysis of the PCR product

14 Extraction of genomic DNA of mycobacteria

15 Extraction of genomic DNA Harvesting the cell Enzymatic treatment phenol-chloroform extraction Purification of the genomic DNA

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17 Amplification of omph gene by PCR

18 Primer sequence : ~Primer 1 (Sense): Corresponding to position 389 to 410 pb

19 ~Primer 2 (antisense): Corresponding to position 1582 to 1604 pb

20 Agarose gel electrophoresis of amplified omph M D:2 A:8 A:5

21 Agarose gel electrophoresis of amplified omph

22 RFLP analysis of omph gene amplified from different strains of P. multocida

23 1 RFLP of PCR product of omph gene of P. Multocida strains using EcoRI.

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27 2 RFLP analysis of PCR product of omph gene of P. Multocida strains using MspI.

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30 3 RFLP analysis of PCR product of omph gene of P. Multocida strains using Hind III

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33 Preparation of omph gene based DNA vaccine

34 Cloning procedures PCR with specific primer Restriction digestion of omph gene and cloning vector Purification of desired fragments ligation Transformation of XL1 Blue E. coli Selection of the +ve colonies

35 Continued Cloning procedures Miniprep Restriction digestion of Miniprep Small scale culture expression SDS-PAGE Western Blotting Maxiprep

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37 Expression cassette in the pbk- CMV phagemid vector

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40 pbk-cmv phagemid multiple cloning site region

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45 Evaluation of the vaccine potential of omph based DNA vaccine against Fowl cholera

46 Evaluation of the vaccine potential of omph based DNA vaccine against Fowl cholera In Mice

47 titer ELISAresults(titer) of mice vaccinated with P. multocida DNA vaccine Titers 0 pre v 1st w p/v 2nd w p/v 3ed w p/v 1st w p/b 2nd w p/b Weeks

48 Titer ELISA results ( titer ) of mive vaccinated with DNA vaccine of P. multocida pre v 1st w p/v 2nd w p/v 3ed w p/v Titers weeks 1st w p/b 2nd w p/b

49 Evaluation of the vaccine potential of omph based DNA vaccine against Fowl cholera In Chicken

50 Experimental Design

51 Group 1: 16 SPF chickens received 100µg of recombinant phagemid Group 2: 16 SPF chickens received 100µg of recombinant phagemid emulsified with incomplete Freund s adjuvant Group 3: 20 SPF chickens received killed P. multocida vaccine. Group 4: 20 SPF chickens were kept without vaccination as negative control.

52 titer Results of IHA of chicken vaccinated with P. multocida DNA vaccine either alone or with freunds and P. multocida killed vaccine P. multocida DNA vaccine alone P. multocida DNA vaccine with Freunds P. multocida killed vaccine 0 prevaccine 1st w P/V 2nd w P/V 3ed w P/V 1st w P/B 2nd w P/B 1st w P/C 2nd w P/C weeks

53 titer ELISA results of chickn vaccinated with P. multocida DNA vaccine either alone or with freunds adjuvent and P. multocida killed vaccine P. multocida DNA vaccine alone P. multocida DNA vaccine with Freunds P. multocida killed vaccine 0 prevaccine 1st w P/V 2nd w P/V 3ed w P/V 1st w P/B 2nd w P/B 1st w P/C 2nd w P/C weeks

54 protection % Results of challange G1 G2 G3 G4 protection % groups

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