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The Construction of Helicobacter Pylori Recombinant Candidate Vaccine Bb-vacA-hpaA

Author: GaoFeng
Tutor: WuLiXian
School: Dali University
Course: Pathogen Biology
Keywords: Helicobacter pylori Bb vacA hpaA vaccine
CLC: R392
Type: Master's thesis
Year: 2011
Downloads: 18
Quote: 0
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Abstract


ObjectiveTo compose the vacA-hpaA fusion gene of Helicobacter pylori; To construct the Escherichia coil-Bifidobacterium shuttle plasmid pGEX-vacA-hpaA; To analyse the plasmid pGEX-vacA-hpaA induced by IPTG in BL21; To transform the recombinant plasmid into Bifidobacterium bifidum by electroporation to construct the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori.MethodsAmplified the vacA and hpaA genes by PCR from plasmids pQE-vacA and pET-hpaA; Constructed recombinant plasmids pQE-hpaA and pQE-vacA-hpaA in order, and amplified the vacA-hpaA fusion gene by PCR from recombinant plasmid pQE-vacA-hpaA. Linked the vacA-hpaA fusion gene to Escherichia coil-Bifidobacterium shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-vacA-hpaA. Introduced the recombinant plasmid pGEX-vacA-hpaA into BL21 by electroporation, analysed the target protein induced by IPTG in BL21 for 4h by SDS-PAGE and Western blot.Finally introduced the recombinant plasmid pGEX-vacA-hpaA into Bb by electroporation to construct the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori.ResultsAgarose gel electrophoresis identified the vacA-hpaA fusion gene was about 1500bp, sequence analysis confirmed the sequence of the vacA-hpaA fusion gene was in accordance with the expected result. Double-digestion demonstrated the vacA-hpaA fusion gene was correctly cloned into the multiple cloning site of Escherichia coil-Bifidobacterium expression shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-vacA-hpaA, and the recombinant plasmid pGEX-vacA-hpaA was successfully introduced into BL21. SDS-PAGE showed the recombinant plasmid pGEX-vacA-hpaA was able to express a 85Kda target protein in E.coil BL21 induced by IPTG for 4h, Western blot showed the target protein could combine specifically with the rabbit VacA immune surum and the rabbit HpaA immune surum. Amplifying the plasmid from the rBb selected in MRS medium with 100μg/ml AMP by PCR and double-digestion demonstrated that the recombinant plasmid pGEX-vacA-hpaA was successfully introduced into Bb and the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori was constructed successfully.Conclusion1. Amplified the vacA-hpaA fusion gene successfully.2. Constructed the recombinant plasmid pGEX-vacA-hpaA successfully.3. The recombinant plasmid pGEX-vacA-hpaA was able to express the target protein in E.coil BL21 induced by IPTG, and the target protein had specific antigenicity of both VacA and HpaA.4. Constructed the Helicobacter pylori recombinant candidate vaccine Bb-vacA-hpaA successfully.

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