Vibrio parahaemolyticus gene deletion attenuated strain, recombinant vibrio parahaemolyticus attenuated live vaccine and preparation method and application thereof
A hemolytic Vibrio, gene deletion technology, applied in the biological field, can solve problems such as affecting health and food safety, drug residues, losses, etc.
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[0100] 6 Preparation of antiserum
[0101] 6.1 Use recombinant attenuated strains to immunize mice. Choose five mice each from the control group and the experimental group. The mice in the experimental group are subcutaneously injected with 106 CFU of recombinant attenuated strains every 7 days. The control group was injected with normal saline. A total of 4 immunizations were performed on the mice
[0102] 6.2 Take blood from mice to prepare antiserum After mice are immunized, insert a needle from the chest below the clavicle, puncture the jugular vein to take blood, and collect the blood into an EP tube. Put the EP tube in a 37°C incubator and place it in a 4°C refrigerator until the blood coagulates, then centrifuge the blood at 4°C and 3500rpm for 15 minutes to carefully absorb the upper layer of serum, and carefully store it at -80°C for future experiments use. At the same time, the serum of the control group was prepared as a control.
Embodiment 1
[0107] Example 1 successfully obtained virulence gene vcrD2 knockout strain (named VP3)
[0108] For the method of obtaining the strains with knockout tdhA and vcrD1 genes, please refer to the master’s thesis published by our laboratory (Zha Zhenzhong, Study on the regulation of host cell immune response by Vibrio parahaemolyticus effector protein VPA1324, Zhejiang Sci-Tech University, 2018).
[0109] 1 PCR amplification of vcrD2 upper and lower homology arm gene sequences and fusion PCR amplification of fusion gene fragments
[0110] According to the vcrD2 (VPA1355) gene sequence in the NCBI database, two primer pairs pRE112-ΔvcrD2-F1 / -R1 and pRE112-ΔvcrD2-F2 / -R2 were used to amplify the upper and downstream homology arm fragments of the gene respectively, and the results showed The two PCR products were respectively at 540bp and 320bp in the agarose gel ( figure 2 a, b). After recovery, use pRE112-ΔvcrD2-F1 / -R2 primers to perform fusion PCR to obtain the fusion fragment o...
Embodiment 2
[0119] Example 2 Successful construction of vector pBBR1MCS-1-ompA-lptD
[0120] 1PCR amplification of the lptD gene fragment and overlapping extension PCR to obtain the ompA-lptD gene fragment
[0121] According to the full gene sequence of lptD and the three-dimensional structure of lptD displayed on NCBI, using the VP bacteria genome as a template, use primers (see Table 2-1) to PCR amplify the 748bp-1343bp gene fragment (corresponding to the 249bp-781bp fragment of the lptD protein) ( Figure 7 a). The DNA sequence coupling (synthesizing) that has lptD fragment and the signal peptide of outer membrane protein OmpA then by overlap extension PCR, the result shows that successfully obtains the lptD gene fragment containing ompA signal sequence ( Figure 7 b). The resulting fusion gene fragment and the pBBR1MCS-1 plasmid need to be digested with the corresponding restriction endonucleases before the next step of the ligation experiment can be performed. The experimental res...
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