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Construction and Expression of the Eukaryotic Expression Vector of Fusion Gene of OprF from Pseudomonas Aeruginosa with HSV-1VP22

Author: YangQin
Tutor: ZhouZhongHui;LeiJun
School: North Sichuan Medical College
Course: Internal Medicine
Keywords: Pseudomonas aeruginosa OprF VP22 DNA vaccine
CLC: R378.991
Type: Master's thesis
Year: 2014
Downloads: 3
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Abstract


Objective: To construct the eukaryotic expression vector for the outermembrane protein F from Pseudomonas aeruginosa and VP22from herpessimplex virus1fusion gene, and express it in eukaryotic cells, which lays thefoundation for the exploiting DNA vaccine of Pseudomonas aeruginosa.Methods: The target gene encoding OprF were amplified by PCR andcloned into the eukaryotic expression vector pVAX1to construct therecombinant plasmid pVAX1-OprF. The target gene encoding VP22wereobtained from the eukaryotic vector pcDNA3-VP22by digestion and clonedinto the eukaryotic expression vector pVAX1to construct the recombinantplasmid pVAX1-VP22. The VP22was inserted into plasmid pVAX1-OprF toconstruct the recombinant plasmid pVAX1-VP22-OprF; The OprF wasinserted into plasmid pVAX1-VP22to construct the recombinant plasmidpVAX1-OprF-VP22. The recombinant plasmids were identified by enzymecutting and sequence analyzing. Meanwhile, the VP22carboxy-terminal wascloned and expressed in E.coli, and purified by electroelution. Polyclonalantibody was developed by immunizing BALB/c mice with purifiedrecombinant protein. The specificity of BALB/c mice antiserum wasmeasured by Western blot. The recombinant plasmids were transfected intoeukaryotic cells by LipofectamineTM. The expressions of recombinanteukaryotic vectors were investigated by Western blot.Results: Digestion and sequence analysis confirmed that the length and the sequence of the fragments inserted were absolutely correct. VP22proteincould be expressed in E.coli BL21(DE3) with high efficiency, obtainedmolecular weight protein of about30kD. Mice immunized with the purifiedprotein produced high specificity of the antibody. The western blot showedthat recombinant vectors could be expression in eukaryotic cells.Conclusion: The recombinant plasmids has been successfullyconstructed. It is demonstrated that the establishment of prokaryoticexpression system and preparation of the polyclonal antibodies could providethe foundation for the future studies. The recombinant plasmids can beexpressed in eukaryotic cells, which lay a solid foundation for further studieson immunological effects of the recombinant plasmids as DNA vaccine.

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Pathogenic bacteria > Other > Blue (green) Pseudomonas aeruginosa
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