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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

Pending Publication Date: 2022-03-08
山东滨州沃华生物工程有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

FAdV-4 mainly causes hydropericardium hepatitis syndrome, which brings serious economic losses to the domestic poultry industry

Method used

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  • 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
  • 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
  • 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

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to the technical field of genetic engineering vaccines, and particularly discloses an I-group 4-type fowl adenovirus fiber-2 protein antigen as well as a method for preparing a genetic engineering subunit vaccine and application of the I-group 4-type fowl adenovirus fiber-2 protein antigen. The method comprises the following steps: adding a non-coding gene in front of a fiber-2 gene of the fowl adenovirus FAdV-4, cloning the non-coding gene to a prokaryotic expression vector pET-28a, transforming the prokaryotic expression vector pET-28a to escherichia coli to construct an engineering bacterium, and inducing the engineering bacterium to express to obtain the soluble fiber-2 protein. Wherein the amino acid sequence of the aviadenovirus fiber-2 antigen protein is SEQ ID NO.2, the nucleotide sequence of a gene coded by the aviadenovirus fiber-2 antigen protein is SEQ ID NO.1, and soluble protein with high expression quantity is obtained through non-coding gene regulation and control. The fiber-2 protein of the fowl adenovirus is expressed through escherichia coli, a soluble expression product is obtained, the subunit vaccine is prepared, safety and effectiveness are achieved, and fowl I-group 4-type adenovirus infection can be prevented.

Description

technical field [0001] The invention relates to the technical field of genetic engineering vaccines, in particular to a fiber-2 protein antigen of group I type 4 avian adenovirus and a method and application thereof for preparing a genetic engineering subunit vaccine. Background technique [0002] Fowl adenovirus (fowl adenovirus, FAdV) belongs to the genus of fowl adenoviridae, fowl adenovirus is divided into three groups, group I, group II and group III. There are 12 serotypes of avian adenovirus group I, which mainly cause inclusion body hepatitis, pericardial effusion and gizzard erosion clinically. Group II avian adenoviruses include turkey hemorrhagic enteritis virus (HEV), marble spleen disease virus (MSDV) and avian adenovirus group II splenomegaly (AASV). Group III adenovirus is mainly egg drop syndrome virus (Eggdrop syn-drom virus, EDSV). FAdV-4 mainly causes hydropericardium hepatitis syndrome, which brings serious economic losses to the domestic chicken indust...

Claims

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Application Information

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IPC IPC(8): C07K14/075C12N15/70C12N15/34A61K39/235A61P31/20
CPCC07K14/005C12N15/70A61K39/12A61P31/20C12N2710/10222C12N2710/10234A61K2039/552A61K2039/5156
Inventor 朱杰王楠杨灵芝王慧李香云王成举
Owner 山东滨州沃华生物工程有限公司
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