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Study on Preparation of Genetic Engineering Live Vector Vaccine of Outer Membrane Protein of Aeromonas Hydrophila and Its Immune Effect in Mice

Author: LiMei
Tutor: LiWeiFen
School: Zhejiang University
Course: Animal Nutrition and Feed Science
Keywords: Aeromonas hydrophila outer membrane protein E.coli Lactococcus lactis expression oral live vector vaccine mice immunity
CLC: S852.5
Type: Master's thesis
Year: 2007
Downloads: 20
Quote: 0
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Abstract


The oral live vector vaccine is popular in recent years because it possesses some advantages, such as simple vaccination route, good immunity and little stress on fishes. Aeromonas hydrophila is a key pathogen in aquaculture. The outer membrane protein of Aeromonas hydrophila, one of the virulence in adhesion, is the general antigen of different serum types. In this study, the gene encoding the outer membrane protein A from Aeromonas hydrophila was cloned and expressed in Escherichia coli and Lactococcus lactis, respectively, and the immunogenicity of the recombinant strains to mice was detected by oral administration. The main results obtained were as follows:1) A pair of clone primers was designed according to the nucleotide sequence encoding outer membrane protein A(ompA) of Aeromonas hydrophila (AF146597). The ompA gene was amplified by PCR using the genome of Aeromonas hydrophila as template. The obtained DNA fragment was cloned into pUCm-T Easy Vector. The result of DNA sequencing showed that the full length of ompA was 1020 bp, encoding 339 amino acids with a signal peptide of 23 amino acids.The molecular weight of precursor protein was 35.9 kDa and the mature protein was 33.7 kDa. The sequence homology with AF146597 was 87.24%at nucleic acid level and 89.2%at amino acid level.2) Two pairs of primers which contain restriction sites SalⅠ/Xho I, EcoRⅠ/Xho I for expression of full length and mature sequence of omp gene in Escherichia coli were designed, respectively. Then the aimed fragments were inserted into the multi clone site (MCS) of pET-30a vector and transformed in E.coli BL21 (DE3). The recombinant strains containing the recombinant plasmid pET-omp1 (full length of ompA) and pET-omp2 (mature length of omp A) were selected and named as E.coli[pET-omp1] and E.coli[pET-omp2]. SDS-PAGE demonstrated that the recombinant outer membrane protein gene was expressed in E.coli BL21 (DE3) successfully.3) Primers introducing restriction sites BamH I and Xba I were designed for expression of mature ompA in Lactococcus lactis. Then the fragment obtained was inserted into the multi clone site (MCS) of pNZ8048 vector and transformed into E.coli MC1061. Then, the recombinant plasmid pNZ8048-omp was transformed Lactococcus lactis by electroporation. A little amount of aimed protein was detected by SDS-PAGE because of its low expression level.4) 120 BALB/c mice were divided into six groups and immunized by intragastric administration with PBS(control), E.coli[pET-omp1], E.coli[pET-omp2], E.coli [pET], L.lactis[pNZ-omp], L.lactis [pNZ] and L.lactis[pNZ-omp] at days1st,2nd,3rd,20th,21st and 22nd. These mice were challenged with Aeromonas hydrophila at day 40th. Results showed that 90%,80%,60%of mice immunized with E.coli[pET-omp1], L.lactis[pNZ-omp], E.coli [pET-omp2] survived, whereas 20%in control group.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Serological
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