Fusion protein antigen of duck hepatitis A virus-3 VP1 protein and LTB and application of fusion protein antigen
A duck hepatitis A virus and fusion protein technology, applied in the field of fusion protein of duck hepatitis A virus VP1 protein and LTB, can solve problems such as ineffective protection.
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Embodiment 1
[0015] The acquisition of the fusion protein of the truncated duck hepatitis A virus VP1 protein and LTB of embodiment 1
[0016] 1. Using the biological software DNAStar to analyze the epitope of the VP1 protein of the representative strain of DHAV-3 (GenBank accession number: EU289393.1), try various technical means such as random splicing of antigen epitopes, connection and integration of antigen epitopes, etc. Finally, the highly antigenic part of the VP1 protein (80-225aa) was selected for splicing with the LTB sequence, and Lingker (GGGS) was designed in the middle for connection to obtain a fusion protein LTB-VP1 of a truncated duck hepatitis A virus VP1 protein and LTB. The amino acid sequence of the encoded protein is SEQ ID NO: 1, and the rare codon optimization of Escherichia coli was performed using the online biological software DNAWorks to obtain the nucleotide sequence SEQ ID NO: 2 of the fusion protein LTB-VP1.
[0017] The nucleotide sequence of the newly obta...
Embodiment 2
[0018] The construction of embodiment 2 genetic engineering protein expression vector and the acquisition of engineering bacteria
[0019] 1. The fusion protein LTB-VP1 gene synthesized by the above-mentioned whole gene was digested with BamH I and Hind III, and then ligated into the corresponding restriction site of the pET30a vector to construct the pET30a / VP1 expression vector.
[0020] 2. Use CaCl 2 The pET30a / VP1 expression vector was transformed into Escherichia coli BL21(DE3), spread on an agar plate containing 50 μg / ml kanamycin, and cultured overnight at 37°C. 10 single colonies were selected to extract plasmids, and the positive colonies verified by BamH I and Hind III double enzyme digestion were further sequenced and identified. The positive clones verified by sequencing were fermented and cultured in LB medium to 0.6-0.8 hours and induced by adding 0.3mMIPTG for 4-5 hours, and the bacteria were collected by centrifugation for SDS-PAGE electrophoresis, and uninduc...
Embodiment 3
[0021] Embodiment 3 fermentation, purification and the preparation of duck hepatitis A virus genetic engineering subunit vaccine
[0022] 1. Fermentation process
[0023] 1) LB medium as seed medium, containing 5g / L glucose and 5g / L MgSO 4 ·7H 2 The LB medium of O was used as the fermentation medium, and the feeding materials were glucose 400g / L, peptone 24g / L, yeast extract 10g / L, NaCl 5g / L, MgSO 4 ·7H 2 O 5g / L.
[0024] 2) Fermentation process
[0025] The identified engineered bacteria were picked and inoculated into LB medium containing kanamycin at a concentration of 50 μg / ml, and cultured with shaking at 37°C for 8 hours as the seed solution. Inoculate the seed liquid in the fermenter according to the inoculum amount of 2%, adjust the parameters, 37°C, 200 rpm, and control the dissolved oxygen above 20%. After 4 hours of fermentation, feed feeding was started, 0.3mmol / L IPTG was added after 6 hours of fermentation to induce expression, and the fermentation ended 6 ...
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