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Recombinant adenovirus for expressing porcine circovirus type-3 ORF2 gene and preparation method and application of recombinant adenovirus

A technology of recombinant adenovirus and gene fragments, which is applied in the field of porcine circovirus type 3 adenovirus vector vaccine, achieving the effects of high yield, low dosage, high immunogenicity and safety

Active Publication Date: 2019-09-20
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no relevant reports on the expression of porcine circovirus type 3 Cap protein and the preparation of adenovirus vector vaccines using the adenovirus expression system. Therefore, the development of PCV3 adenovirus vector vaccines is of great significance

Method used

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  • Recombinant adenovirus for expressing porcine circovirus type-3 ORF2 gene and preparation method and application of recombinant adenovirus
  • Recombinant adenovirus for expressing porcine circovirus type-3 ORF2 gene and preparation method and application of recombinant adenovirus
  • Recombinant adenovirus for expressing porcine circovirus type-3 ORF2 gene and preparation method and application of recombinant adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of a recombinant adenovirus transfer vector carrying the Cap protein coding gene of porcine circovirus type 3

[0042] 1. Codon optimization and synthesis of the Cap protein coding gene of PCV3

[0043] With reference to the sequence of the Cap protein coding gene (ORF2 gene) of PCV3 in the database, in order to achieve high-efficiency expression of the Cap protein, the present invention first optimizes the ORF2 gene sequence according to the codon preference of the adenovirus expression system, and designs different The codon optimization sequence of the codon, and the preliminary detection of its expression level. After comparison and screening, the present invention found that the expression level of the coding sequence obtained by different degrees of codon optimization is quite different, and the codon optimization position and degree of optimization and expression There is no clear rule between levels. For example, the expression level of the cod...

Embodiment 2

[0064] Example 2 Construction of recombinant adenovirus Ad-PCV3 Cap

[0065] 1. Construction of recombinant adenovirus plasmid pAd-PCV3 Cap

[0066] The recombinant plasmids pShuttle-CMV-Cap-1, pShuttle-CMV-Cap-2 and pShuttle-CMV-Cap-3 were linearized with PmeI, and the digested products were recovered and transformed into E.coli containing the backbone vector pAdEasy-1 BJ5183 competent cells. Pick the positive clones containing the recombinant adenovirus plasmids pAd-PCV3 Cap-1, pAd-PCV3 Cap-2 and pAd-PCV3 Cap-3, and extract the plasmids after shaking. The recombinant adenovirus plasmids pAd-PCV3 Cap-1, pAd-PCV3 Cap-2 and pAd-PCV3 Cap-3 were digested and identified with PacⅠ. The identification results are as follows image 3 Shown, and sent for sequencing verification.

[0067] Obtain a large number of pAd-PCV3 Cap recombinant plasmids: Take 2μl of recombinant transfer plasmids pAd-PCV3 Cap-1, pAd-PCV3 Cap-2, and pAd-PCV3 Cap-3 into 100μl of E. coli competent DH10B. After 30 minu...

Embodiment 3

[0075] Example 3 Evaluation of safety and immunogenicity of adenovirus Ad-PCV3 Cap vector vaccine

[0076] 1. Safety evaluation of adenovirus Ad-PCV3 Cap vector vaccine in mice

[0077] Purchase 10 female Balb / C mice of 16-18g and divide them into two groups A and B with 5 mice in each group. Each mouse in group A was injected subcutaneously with 10 8 The recombinant adenovirus Ad-PCV3 Cap-1 prepared in Example 2 of TCID50; each mouse in group B was injected subcutaneously with 0.1mL sterile PBS and observed for 14 days. The two groups of mice were in the same state and there was no abnormal reaction. This result It shows that the adenovirus vector vaccine prepared from the recombinant adenovirus of Example 2 is safe for mice.

[0078] 2. The adenovirus Ad-PCV3 Cap vector vaccine was evaluated for immunogenicity in mice

[0079] Purchase 10 female Balb / C mice aged 6-8 weeks and randomly divide them into two groups A and B, with 5 mice in each group. Each mouse in group A was injecte...

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Abstract

The invention relates to the field of biotechnology, in particular to a recombinant adenovirus for expressing porcine circovirus type-3 ORF2 gene and a preparation method and application of the recombinant adenovirus. The invention provides a recombinant adenovirus transfer plasmid containing a Cap protein coding gene of PCV3 and a recombinant adenovirus. According to the invention, through artificial codon optimization and signal peptide selection, high-level secretion expression of the Cap protein of PCV3 is realized, the structure and function of the obtained Cap protein of PCV3 are completely consistent with the natural state, the immunogenicity of the Cap protein is reserved to the greatest extent, and the Cap protein can be recognized by antigen presenting cells such as DC better and faster. The adenovirus vector vaccine provided by the invention has high immunogenicity and safety, can effectively activate the immune response of an organism, stimulates animals to generate high-level specific antibodies and neutralizing antibodies, and plays an excellent immune protection effect on porcine circovirus.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant adenovirus expressing porcine circovirus type 3 ORF2 gene and a preparation method thereof, and a porcine circovirus type 3 adenovirus vector vaccine prepared by using the recombinant adenovirus. Background technique [0002] Porcine Circovirus (Porcine Circovirus, PCV) is a single-stranded circular DNA virus. It is one of the smallest animal DNA viruses. It can cause piglet multi-system failure syndrome, pig respiratory mixed disease, pig reproductive disorder, and pig dermatitis nephropathy. Syndrome, etc., cause serious harm to the pig industry. Two types of porcine circovirus were identified early, including circovirus type 1 (PCV1) and circovirus type 2 (PCV2). In 2015, an outbreak of dermatitis and nephropathy occurred in a herd of sows on a pig farm in the United States. The clinical symptoms and histological changes were consistent with circovirus-related diseases, ...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/34C12N7/01A61K39/12A61P31/20
CPCA61K39/12A61P31/20C07K14/005C12N15/86C12N2710/10021C12N2710/10043C12N2750/10022C12N2750/10034C12N2800/22
Inventor 张华伟徐高原朱娴静周明光陈波郝根喜方玉林
Owner WUHAN KEQIAN BIOLOGY CO LTD
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