Preparation method and application of haemophilus paragallinarum trivalent genetic engineering subunit vaccine

A vaccine and gene technology, applied in the field of preparation of Haemophilus paragallinarum trivalent genetic engineering subunit vaccine, can solve the problems affecting vaccine safety, cumbersome purification process, serious side effects, etc., achieve superior immune effect and ensure immune effect , the effect of increasing the antibody level

Active Publication Date: 2022-04-01
扬州优邦生物药品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is that the whole bacteria vaccine used to prevent and treat chicken infectious rhinitis has the problems of high cost and serious side effects, and when preparing the genetic engineering subunit vaccine, the antigen obtained by using the Escherichia coli expression system needs to be tediously processed Purification process, difficult to industrialize and affect vaccine safety

Method used

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  • Preparation method and application of haemophilus paragallinarum trivalent genetic engineering subunit vaccine
  • Preparation method and application of haemophilus paragallinarum trivalent genetic engineering subunit vaccine
  • Preparation method and application of haemophilus paragallinarum trivalent genetic engineering subunit vaccine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Construction of recombinant baculovirus rBac-rApg-ABC

[0025] 1. Design the gene fragment encoding the fusion protein rApg-ABC, and submit it to Nanjing GenScript for sequence optimization according to the codon preference of insect cells, and synthesize the optimized sequence (SEQ ID NO:2) onto the pFastBac I transfer vector , to obtain the pFastBac-rApg-ABC transfer vector.

[0026] 2. Construction of recombinant baculovirus:

[0027] The transfer vector pFastBac-rApg-ABC was transferred into Escherichia coli DH10Bac competent cells, and positive clones were selected for PCR identification with M13 primers. M13-F: TGTAAAACGACGGCCAGT; M13-R: CAGGAAACAGCTATGAC. The PCR reaction system was (total volume 25 μL): 0.5 μL DNA template, 0.5 μL each of M13-F and M13-R, 12.5 μL DNA polymerase and 11 μL sterile water. The PCR reaction conditions are: 95°C, 5min; 30 cycles of 95°C for 30s, 65°C for 30s, 72°C for 90s; 72°C for 10min. 1% agarose gel electrophores...

Embodiment 2

[0029] Embodiment 2: Preparation of recombinant rApg-ABC protein

[0030] 1. Amplification of recombinant baculovirus: inoculate insect cell sf9 with recombinant baculovirus rBac-rApg-ABC, culture at 27°C for 4 days, collect the culture, centrifuge and take the supernatant to obtain the f2 generation recombinant baculovirus.

[0031] 2. Identification of expressed proteins

[0032] (1) The f2 generation recombinant baculovirus was inoculated into insect cell sf9 at an inoculum amount of MOI=5-10, cultured at 27° C. for 4 days, the culture was collected, and the supernatant was collected by centrifugation to obtain the recombinant rApg-ABC protein.

[0033] (2) SDS-PAGE identification: The above supernatant was subjected to SDS-PAGE electrophoresis; after electrophoresis, after staining and decolorization, it was found that there was a band at about 66kDa, and its molecular weight was consistent with the theoretical size, indicating that the expression was successful.

[0034]...

Embodiment 3

[0038] The preparation of embodiment 3 Haemophilus paragallinarum trivalent genetic engineering subunit vaccine

[0039] Take the rApg-ABC recombinant protein obtained in Example 2, add an adjuvant to emulsify, mix well, and store at 4°C. See Table 1 for specific vaccine ratios.

[0040] Table 1 Composition ratio of duck Tembusu virus genetically engineered subunit vaccine

[0041]

[0042] Note: ※ represents the protein concentration in each ml of vaccine.

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Abstract

The invention discloses a preparation method and application of a haemophilus paragallinarum trivalent genetic engineering subunit vaccine, and belongs to the field of veterinary biological products. According to the invention, the fusion protein rApg-ABC capable of simultaneously preparing three serotypes of haemophilus paragallinarum A, B and C is constructed and expressed. According to the invention, an rApg-ABC fusion protein gene is connected to a pFastBac1 vector, and the rApg-ABC fusion protein gene is transformed into a DH10Bac competent cell to obtain a recombinant rod grain. And transfecting the recombinant baculovirus to sf9 cells, culturing to obtain recombinant baculovirus, inoculating the recombinant baculovirus into suspended insect cells HF to efficiently express antigen protein, extracting, purifying, carrying out BEI inactivation, adding an adjuvant, and emulsifying to obtain the vaccine. The preparation method is simple, can prepare a large amount of antigen protein, is short in time consumption and high in expression quantity, greatly reduces the production cost, and is beneficial to large-scale production.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a preparation method and application of a trivalent genetic engineering subunit vaccine of Haemophilus paragallinarum. Background technique [0002] Haemophilus paragallinarum (Haemophilus paragallinarum, Hpg), is a short Gram-negative bacillus of Pasteurellaceae Avibacterium (Avibacterium), is the pathogenic bacterium of chicken infectious coryza (Infectious coryza, IC), causing acute or sub Acute respiratory infection, the clinical manifestations are nasal cavity, infraorbital sinus and upper trachea inflammation, tearing, nasal discharge, and dyspnea. Sick chickens sneeze, shake their heads, and have edema on one or both sides of the face. Haemophilus paragallinarum can cause a decrease in egg production of laying hens, a decrease in broiler quality, growth retardation of growing flocks and an increase in culling rate, causing large economic losses to...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N7/01C12N15/62C07K19/00A61K39/102A61K39/116A61P31/04C12R1/93
CPCY02A50/30
Inventor 潘杰李晓明迟强伟王绍君丁国伟荣雪路李甜甜魏荣荣叶正琴李琛潘晨陈林中日陈森
Owner 扬州优邦生物药品有限公司
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