Anti-tetrodotoxin single-chain antibody and preparation method thereof
A technology of tetrodotoxin and single-chain antibody, which is applied to the preparation method of peptide, biochemical equipment and method, chemical instrument and method, etc., and can solve the problem of large relative molecular mass of antibody
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Embodiment 1
[0035] Example 1 Anti-tetrodotoxin single chain antibody heavy chain variable region gene (V H ) and the light chain variable region gene (V L ) clone
[0036] 1) The cells used are mouse-derived anti-tetrodotoxin antibody monoclonal cells
[0037] The cells used were hybridoma cell line Escherichia coli DH5α B10 with anti-tetrodotoxin monoclonal antibody.
[0038] The hybridoma cell line Escherichia coli (Escherichia coli) DH5α B10 was routinely cultured in RPMI1640 medium with 10% fetal bovine serum at 37°C, 5% CO 2 to cultivate.
[0039] 2) Primer design
[0040] The variable region gene sequence of the antibody to be amplified is unknown, so the primers used in the present invention adopt the internationally popularized primer sequences.
[0041] at V L The 5' end of the primer was added with a restriction site Nco Ⅰ: CATGCCATGG (including the protective base CATG), at the V H A restriction site Xho I: CCGCTCGAG (including the protective base CCG) was added to the 3...
Embodiment 2
[0058] Example 2 Cloning of anti-tetrodotoxin single chain antibody gene and construction of its expression vector
[0059] to recover the V H and V L The gene product mixture is used as a template, and the V H 5' and V L The 3' primer amplifies the anti-tetrodotoxin single-chain antibody gene (V L -Linker-V H ), and then amplify the anti-tetrodotoxin single-chain antibody gene by expression vector primers (see figure 2 ), its insertion expression vector sequence is:
[0060]
[0061]
[0062] Among them, the 1st to 438th nucleotides are the light chain variable region gene of the anti-tetrodotoxin single-chain antibody, with a total length of 438bp; the 439th to 483rd nucleotides are the connecting peptide Linker, with a total length of 45bp; 484th to 858th The first nucleotide is the heavy chain variable region gene of the anti-tetrodotoxin single chain antibody, and the full length is 375bp.
[0063] The PCR reaction system is: 10×PCR buffer 2.5 μL; dNTP (10 m...
Embodiment 3
[0067] Example 3 Prokaryotic expression and purification of anti-tetrodotoxin single chain antibody gene
[0068] Select a single colony of E.coli BL21(DE3) containing the pET-22b(+)-scFv recombinant plasmid with correct sequencing, culture with shaking at 37°C overnight, dilute it into LB culture medium at 1:100, and culture with shaking until OD 600 When the value is about 0.6 to 0.8, add IPTG with a final concentration of 0.5mmol / L to induce expression for 4 hours, collect the bacteria for SDS-PAGE electrophoresis, and after staining and decolorization, it is found that the expression product of E.coli BL21 (DE3) has The obvious band, consistent with the size of the expected target protein band, indicates that the anti-tetrodotoxin scFv gene has been expressed (see image 3 ). The amino acid sequence of the anti-tetrodotoxin single chain antibody is:
[0069]
[0070]
[0071] SDS-PAGE electrophoresis showed that most of the prokaryotic expression products of the an...
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