Duck hepatitis A virus VP1 protein gene and application thereof
A technology of duck hepatitis A virus and gene, applied in the direction of antiviral immunoglobulin, application, antiviral agent, etc., can solve the problems of inability to protect duck viral hepatitis diseases
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Embodiment 1
[0015] Embodiment 1 The acquisition of novel composite duck hepatitis A virus VP1 protein and its gene
[0016] 1. Carry out antigenicity analysis on the VP1 protein sequence of the representative strain of DHAV-1 (GenBank accession number: GU363950.1) and the VP1 protein sequence of the representative strain of DHAV-3 (GenBank accession number: EU289393.1), and select DHAV respectively -1 VP1 protein dominant epitope (94-146) is spliced with DHAV-3 VP1 protein dominant epitope (115-223), and protein lingker (GGGSGGGS) is added in the middle to promote the correct folding of the two sequences, and finally The amino acid sequence of the obtained novel composite duck hepatitis A virus VP1 protein is SEQ ID NO:1.
[0017] 2. According to the obtained amino acid sequence SEQ ID NO: 1 of the novel composite duck hepatitis A virus VP1 protein, redesign according to the codon preference of the Escherichia coli gene, and obtain the nucleotide sequence SEQ ID NO: 2 encoding the compo...
Embodiment 2
[0018] The construction of embodiment 2 genetic engineering protein expression vector and the acquisition of engineering bacteria
[0019] 1. The VP1 gene protein gene BamH I and Hind III synthesized by the above-mentioned whole gene was double digested and then ligated into the corresponding restriction site of the pET28a vector to construct the pET28a / VP1 expression vector.
[0020] 2. Use CaCl 2 Transform the pET28a / VP1 expression vector into Escherichia coli BL21(DE3), spread it on an agar plate containing 50 μg / ml kanamycin, and culture 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 0.5mM IPTG for 4-5 hours, and the bacteria were collected by centrifugation for SDS-PAGE electrophoresis, and uninduced bacte...
Embodiment 3
[0021] Example 3 Fermentation, purification and preparation of compound duck hepatitis A virus genetically engineered 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. The seed liquid is inoculated in the fermenter according to the inoculation amount of 5%, and all parameters are adjusted, 37°C, 200 rpm, and the dissolved oxygen is controlled above 20%. After 4.5 hours of fermentation, feed feeding was started, 0.5mmol / L IPTG was added after 6 hours of fermentation to induce expression,...
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