A kind of polypeptide for preparing fish immune adjuvant and its application
An immune adjuvant, a technology of use, applied in the field of immunology, to achieve the effect of convenient use, simple preparation, and enhanced immune protection effect
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Embodiment 1
[0052] The preparation of embodiment 1 polypeptide of the present invention
[0053] 1. Preparation of the gene of the present invention
[0054] Design primers: the 5' end of the upstream primer (P1) and the 5' end of the downstream primer (P2) plus enzyme cutting sites EcoR Ⅰ and Xho Ⅰ, the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., the sequence is as follows:
[0055] P1 5'-CG GAATTC GGAGAAGGAAAAGCAGAG-3' (the underlined part is the EcoR Ⅰ restriction site);
[0056] P2 5'-CC CTCGAG TCAGTTTTGCTGTTTGATCT-3' (the underlined part is the Xho I restriction site).
[0057] Using channel catfish spleen cDNA as a template and P1 and P2 as primers, the IL-8 gene of the present invention (abbreviated as mCcIL-8) was amplified by PCR using a 25 μL reaction system, as shown in Table 1.
[0058] Table 1 The PCR amplification system of the gene of the present invention
[0059]
[0060] PCR reaction conditions: pre-denaturation at 95°C for 5 minutes...
Embodiment 2
[0090] The preparation of embodiment 2 subunit vaccine of the present invention
[0091] 1. Antigen preparation
[0092] Using channel catfish spleen cDNA as template, P3 and P4 as primers,
[0093] P3: 5'-CGCGGATCCATGTCAATTATTACTGATG-3' (the underlined part is the BamHI restriction site);
[0094] P4: 5'-CGGAAGCTTCTATTTTTTTAGGTTGTAG-3' (the underlined part is the HindIII restriction site);
[0095] A 1308bp S.iniaeа-enolase gene was amplified;
[0096] The nucleotide sequence of the S. iniae а-enolase gene is shown below:
[0097]
[0098] Insert the S.iniae а-enolase gene into pET32a(+) to obtain pET32-eno, and further transfer it into BL21(DE3) competent cells to obtain recombinant strains;
[0099] The recombinant strain was induced with 0.1mM IPTG at 37°C for 4h, and the cells were collected by centrifugation. The supernatant was collected after the cells were ultrasonically disrupted, and purified according to the operating instructions of the Ni-NTA-Sefinose Col...
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