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Novel genetic engineering subunit vaccine for avian Newcastle disease viruses

A technology of Newcastle disease virus and coding gene, which is applied in the field of new genetic engineering subunit vaccine of avian Newcastle disease virus, can solve problems such as incomplete inactivation, and achieve the effect of shortening protein purification time, reducing complexity and facilitating large-scale production.

Active Publication Date: 2020-05-15
苏州沃美生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inactivation process may have the risk of incomplete inactivation

Method used

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  • Novel genetic engineering subunit vaccine for avian Newcastle disease viruses
  • Novel genetic engineering subunit vaccine for avian Newcastle disease viruses
  • Novel genetic engineering subunit vaccine for avian Newcastle disease viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Embodiment 1: Construction of recombinant eukaryotic expression vector pCI-HN-MC-NT-GS

[0136] 1. HN-MC-NT gene amplification and purification The codon-optimized HN protein coding gene (can be defined as HN-MC-NT gene, HN represents the extracellular region of NDV HN protein, MC represents the C-terminal fragment of M protein, NT stands for N protein T cell epitope) from Nanjing GenScript Biotechnology Co., Ltd., and cloned into the pUC-57 vector to construct the pUC-HN-MC-NT plasmid vector. The optimized HN-MC-NT gene sequence is shown in SEQ ID NO:1. Using pUC-HN-MC-NT as a template, and HN-F and HN-R as primers for PCR amplification (the gene sequences of HN-F and HN-R are shown in SEQ ID NO.5 and 6), the amplification system See Table 1. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes, and st...

Embodiment 2

[0151] Embodiment 2: Construction of recombinant eukaryotic expression vector pCI-F-MC-NT-GS

[0152] 1. F-MC-NT Gene Amplification and Purification Nanjing GenScript Biotechnology Co., Ltd. synthesized a codon-optimized F protein-encoding gene (which can be defined as the F-MC-NT gene, and F represents the extracellular region, MC represents the C-terminal fragment of M protein, and NT represents the T cell epitope of N protein), and cloned into the pUC-57 vector to construct the pUC-F-MC-NT plasmid vector. The optimized F-MC-NT gene sequence is shown in SEQ ID NO:3. Using pUC-F-MC-NT as a template and F-F and F-R as primers for PCR amplification (the gene sequences of F-F and F-R are shown in SEQ ID NO.7 and 8), the amplification system is shown in Table 5. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes...

Embodiment 3

[0166] Example 3: Construction and screening of recombinant CHO cells expressing HN-MC-NT protein and F-MC-NT protein

[0167] 1. Cell Transfection

[0168] 1.1 Prepare cells Take CHO cells in the logarithmic growth phase, sample and count, and use 1×10 6 The cell density of cells / ml continues to be subcultured, maintain the seeds, centrifuge the remaining cells, centrifuge at 1000rpm for 4 minutes, discard the supernatant, resuspend with about 20ml of fresh CHO-WM medium, centrifuge again, centrifuge at 1000rpm for 4 minutes, discard the supernatant After resuspending with a small amount of medium for counting, the final cell density was adjusted to 1.43×10 7 cells / ml.

[0169] 1.2 Plasmid and cell mixing Take 5 μg of the pCI-HN-MC-NT-GS plasmid vector in Example 1, add it to the EP tube, add 0.7ml cells, mix well, and let stand for 15 minutes.

[0170] 1.3 Electroporation 280V20ms electric shock for 2 pulses, after the electric shock is completed, immediately transfer the...

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Abstract

The invention provides a novel genetic engineering subunit vaccine for avian Newcastle disease viruses. The preparation method for the vaccine comprises the following steps: optimizing coding genes ofHN protein and F protein of the avian Newcastle disease viruses (NDV); respectively cloning eukaryotic expression vectors containing optimized HN and F protein coding genes, respectively transfectingCHO cells, screening CHO cell strains capable of stably and efficiently expressing recombinant HN and F proteins in a suspension manner, culturing the CHO cell strains, and carrying out separation soas to obtain recombinant HN and F proteins; and fully and uniformly mixing the recombinant HN protein, the recombinant F protein and an adjuvant. According to the invention, coding genes of importantantigen proteins HN protein and F protein of NDV are optimized; CHO cells are used for eukaryotic expression; protein glycosylation is sufficient; antigen protein immunogenicity is good; safety is high; the expression quantity can reach 2-4 g / L; meanwhile, recombinant cells can be subjected to large-scale suspension culture; the complex degree of vaccine preparation is greatly reduced; the process controllability is good; the quality of products between batches is stable; and the production cost is remarkably reduced.

Description

technical field [0001] The application relates to the technical field of animal immunization drugs, in particular to a novel genetically engineered subunit vaccine of avian Newcastle disease virus. Background technique [0002] Avian Newcastle Disease (ND) is an acute highly contagious disease caused by avian Newcastle Disease Virus (NDV). The disease can occur throughout the year, especially in cold and climate-changing seasons. The main features of the disease are dyspnea, neurological disturbance, mucosal and serosal hemorrhage and necrosis. Avian Newcastle disease has a high morbidity and fatality rate, and it occurs frequently all over the world. Once it breaks out, it will bring a devastating blow to the poultry industry. It is a major infectious disease that endangers the poultry industry. OIE lists it as a Category A disease. The incidence of ND in my country is very high, and the mortality rate is as high as more than 90%, which seriously endangers the healthy de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/45C07K14/125A61K39/17A61P31/14
CPCA61K39/12A61K2039/552A61P31/14C07K14/005C12N2760/18122C12N2760/18134
Inventor 曹文龙孔迪滕小锘易小萍张大鹤
Owner 苏州沃美生物有限公司
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