A kind of eel pathogenic bacteria single-chain antibody and its application
A single-chain antibody and pathogenic bacteria technology, applied in the fields of applications, bacteria, fungi, etc., can solve the problems of artificial active immune adaptability limitations, long process time, etc., to improve the level of cellular immunity, high inhibition rate, and reduce death rate effect
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Embodiment 1
[0040] Example 1 Construction of eel immunoglobulin single-chain T7 phage antibody library and screening and detection of anti-recombinant outer membrane protein specific antibodies
[0041] The cDNAs encoding eel IgH and IgL were cloned and analyzed by molecular cloning methods such as RT-PCR and RACE, respectively, and the VH and VL antibody gene libraries were amplified according to the gene sequences of the heavy chain and light chain variable regions. Methods The heavy chain variable region gene and light chain variable region gene were spliced into a single-chain antibody (ScFv) gene library by overlap extension reaction, and then connected with the T7 phage vector to construct the eel T7 phage antibody library. The constructed T7-Scfv phage antibody library was used to screen the recombinant outer membrane protein single-chain antibody of Aeromonas hydrophila with immune cross-protection. After 4 rounds of panning of the antibody library, 10 strains of antibody plaque...
Embodiment 2
[0119] Example 2 Prokaryotic expression and eukaryotic expression of specific anti-outer membrane protein single chain antibody
[0120] The specific antibody gene OMP-Scfv obtained by screening was used as a cloning template, and prokaryotic expression and eukaryotic expression were performed on it. Select pGEX-4T-2 as the prokaryotic expression vector, select ECOR I and NOT I as restriction sites according to the target gene and the plasmid map of the expression vector, and add a HIS tag between the target gene at the 3' end and the restriction site when designing primers , in order to prepare for the subsequent purification experiment, after the recombinant vector pGEX-4T-2-OMP was established, the bacterial cells obtained by sequencing were induced and expressed using IPTG as an inducer, and the expressed protein in the presence of inclusion bodies was subjected to centrifugal Ni affinity chromatography The column was purified, and urea-PBS solution with gradient concentra...
Embodiment 3
[0192] Example 3 Injection of single-chain antibody and recombinant outer membrane protein on eel immune protection
[0193] 1. Materials and methods
[0194] 1.1 Experimental animals
[0195] The purchased 1000 European eels (Anguilla anguilla), weighing about 50g, were divided into 6 groups, 150 in each group, and bred in the breeding test field of the Fisheries College, of which 100 were used to detect the immune protection effect after challenge, and the rest were used for The levels of various immune indicators were measured, the water temperature was 23-28°C, sufficient oxygen was maintained, feeding and excrement were removed on time every day, and the animals were kept for 1 week.
[0196] 1.2 Strain culture
[0197] The Aeromonas hydrophila (B11, expressing the recombinant outer mold protein OMP, whose amino acid sequence is shown in SEQID NO 05) kept in the laboratory was inoculated on Tryptone Soy Agar (TSA) Tryptone Soy Broth (TSB), Cultivate overnight at 28°C; ...
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