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Recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene, recombinant baculovirus and preparation method and application thereof

A recombinant baculovirus, porcine pseudorabies technology, applied in the direction of virus/phage, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of not carrying a single gene of PRV, and achieve strong diagnostic antigen specificity. , The effect of high protection rate and high sensitivity

Active Publication Date: 2019-06-28
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Subunit vaccines carrying a single gene of PRV have not yet appeared

Method used

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  • Recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene, recombinant baculovirus and preparation method and application thereof
  • Recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene, recombinant baculovirus and preparation method and application thereof
  • Recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene, recombinant baculovirus and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Construction method of recombinant baculovirus transfer vector PFBD-2-PRVgD comprising porcine pseudorabies virus gD protein gene

[0085] The coding gD with the deletion of the transmembrane region fragment, the gD is connected with the pEASYBLUNT vector to obtain the pEASYBLUNT-gD recombinant vector, and then verified by sequencing, and the result is correct after comparison.

[0086] Using the plasmid pEASYBLUNT-gD as a template, two sets of primer pairs were used to perform PCR amplification to obtain the gD and gD genes connected with the GP67 signal peptide sequence, and then clone the target gene into the baculovirus transfer by BamHI, EcoRI, XbaI and HindIII, respectively. The vector PFBDHmHNM1P10eEFP is referred to as PFBD (see Chinese patent 200910063217.6, a recombinant baculovirus expressing a modified and synthesized sandwich H1N1 influenza virus HA-NA-M1 gene). Specifically, using pEASYBLUNT-gD as a template, use the first primer pair to perform PCR amplif...

Embodiment 2

[0091] Construction method of recombinant baculovirus AC-2-PRVgD

[0092] Take 1 μg of the baculovirus transfer plasmid PFBD-2-PRV-gD and add it to DHI0Bac Escherichia coli competent cells, and ice-bath for 30 minutes. Heat shock in water at 42°C for 45 seconds, then ice-bath for 2 minutes, add 900 microliters of SOC liquid medium, shake and culture at 37°C for 4 hours, take 100 microliters and spread on three-antibody LB plates (Kan, Gen and Tel), Cultivate at 37°C for 24-48 hours. Pick a single white colony that has passed three rounds of blue-white screening and place it in 10 mL of LB medium containing appropriate concentrations of kanamycin, gentamicin, and tetracycline, and culture it on a shaker (180 rpm / min) at 37°C for 16 hours. Collect bacteria by centrifugation, add 0.3mL Solution I to resuspend, add 0.3mL Solution II to mix gently, immediately add 0.3mL Solution III to mix, ice bath for 10min, centrifuge at 12000rpm at 4°C for 10min, transfer the supernatant to an...

Embodiment 3

[0095] Artificially synthesize the nucleotide sequence gD encoding the gD protein and the IgG2aFc encoding the nucleotide sequence of the mouse IgG2aFc protein with the deletion of the C-terminal transmembrane region fragment; the volume ratio of the gD or IgG2aFc to the pEASYBLUNT vector is 0.5-4:1 The ratio was mixed and reacted at room temperature for 5 minutes, and the pEASYBLUNT-gD and pEASYBLUNT-IgG2aFc recombinant vectors were obtained by ligation, and the sequencing was verified, and the results were correct after comparison.

[0096] Using pEASYBLUNT-gD as a template, use the first primer pair in Example 1 to carry out PCR amplification respectively to obtain the target fragment connected with the HBM signal peptide-gD-His; the product HBM-gD-His obtained by amplifying the first primer pair and pEASYBLUNT-IgG2aFc as a template, and use the second primer pair to perform PCR amplification respectively to obtain the HBM signal peptide-gD-IgGFc-His target fragment connecte...

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Abstract

The invention provides a recombinant baculovirus transfer vector containing a porcine pseudorabies virus gD protein gene, a recombinant baculovirus and a preparation method and application thereof, and belongs to the technical field of veterinary vaccines. The recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene is prepared by inserting HBM signal peptide-gD-His tagor HBM signal peptide-gD-IgGFc-His tag as an exogenous gene into an enzyme cutting site between BamHI and EcoRI and an enzyme cutting site between SpeI and HindIII of the baculovirus transfervector respectively; and the gD is a gD protein gene deleted from a encoding transmembrane region fragment. The vector promotes the secretory expression of the porcine pseudorabies virus gD protein and the gD-IgGFc fusion protein. The subunit vaccine prepared by using the gD protein and the gD-IgGFc fusion protein has the advantages of good safety, strong specificity and high virus attack protection rate.

Description

technical field [0001] The invention belongs to the technical field of veterinary vaccines, and in particular relates to a recombinant baculovirus transfer vector containing porcine pseudorabies virus gD protein gene, a recombinant baculovirus, a preparation method and an application. Background technique [0002] Porcine pseudorabies (Pseudorabies, PR also known as Aujeszky's disease, AD) is an infectious disease caused by porcine pseudorabies virus (Pseudorabies Virus, PRV). Except that pigs and cattle of all ages are susceptible, sheep, dogs, cats, rabbits, rats, minks, foxes and other animals are infected and diseased under natural conditions, which can easily cause large economic losses to the animal husbandry industry. Porcine pseudorabies virus (Pseudorabies virus, PRV), also known as porcine herpesvirus type Ⅰ, infectious bulbar paralysis virus, scrapie virus, Oyezki's disease virus, can easily cause various diseases such as cattle, sheep, pigs, dogs and cats. Sympt...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/66C12N15/38C12N7/01A61K39/245A61P31/22
Inventor 钱平李祥敏李江龙陈焕春
Owner HUAZHONG AGRI UNIV
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