Group I type 4 fowl adenovirus fiber-2 protein antigen as well as method for preparing genetic engineering subunit vaccine and application of group I type 4 fowl adenovirus fiber-2 protein antigen
A fiber-2, subunit vaccine technology, applied in genetic engineering, viral antigen components, botanical equipment and methods, etc., can solve problems such as economic losses in the domestic chicken industry, achieve good commercial development prospects, and prevent type 4 Effects of adenovirus infection
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Embodiment 1
[0036] Example 1: Amplification of fiber-2 gene
[0037] According to the avian adenovirus fiber-2 gene sequence published in NCBI, the primer sequences were designed and synthesized as follows:
[0038] fiber2-f: 5'-CCCGAATTCACGATCGCAGCGATGCTCGGGGACCCTATAAGAAGTC-3',
[0039] fiber2-r: 5'-CTAAAGCTTTTACGAGAGGCAGGCCTCTGGTCAG-3'.
[0040] The suspected pericardial effusion syndrome disease materials were collected from a chicken farm, and adenovirus infection was diagnosed by laboratory testing, and the adenovirus was successfully isolated.
[0041] Using the isolated adenovirus nucleic acid as a template, the fiber-2 gene was amplified with fiber2-f and fiber2-r primers, and the amplified product was cloned into the pMD-18T vector to obtain pMD-18T-fiber2, which was sent to Shanghai Sangong for assay Sequence, the fiber-2 nucleotide sequence is SEQ ID NO.1, and the corresponding amino acid sequence is SEQ ID NO.2.
Embodiment 2
[0042] Embodiment 2: Construction of expression plasmid
[0043] 2.1 Enzyme digestion reaction
[0044] Both the pMD-18T-fiber2 plasmid and pET-28a were double-digested with EcoR I and Hind III, 50 μL double-digested and the reaction was as follows:
[0045] Table 1
[0046]
[0047] Place the enzyme digestion system in a 37°C water bath for 1 hour to recover target fragments and vectors.
[0048] 2.2 Purpose Fragment Recovery
[0049] The digested products were subjected to 1% agarose gel electrophoresis, and the gel recovery kit was used according to the instructions to recover the target fragment of about 1440 bp in the pMD-18T-fiber2 plasmid and the 5900 bp fragment in the pET-28a plasmid respectively.
[0050] 2.3 Ligation reaction
[0051] Ligate the recovered target fragment and plasmid according to the T4 DNA ligase reaction, and the 20 μL ligation system is as follows:
[0052] Table 2
[0053]
[0054] The connection system was reacted at 25°C for 30 minute...
Embodiment 3
[0058] Example 3: Induced expression and identification of fiber-2 protein fragments
[0059] The expression strain containing pET28a-fiber2 prepared in Example 2 was inoculated into LB medium containing 50 μg / ml kanamycin according to a ratio of 1% (v / v), and the bacteria were shaken at 37°C and 220rpm until When the OD600 is between 0.6-0.8, add 0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG), shake and induce overnight at 28°C, and collect the bacteria by centrifugation after the culture is over. Sludge was reconstituted with PBS, crushed, and SDS sample buffer was added to the supernatant and precipitate respectively, boiled for 10 minutes, and polyacrylamide gel electrophoresis was used to find that the fiber-2 protein was mainly soluble expressed protein.
[0060] The SDS-PAGE gel obtained in the previous step was transferred, blocked, washed 3 times with PBST, incubated with 1:200 diluted primary antibody (poultry adenovirus positive serum), washed 3 times with PBST, 1...
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