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Celine parvovirus-like particle as well as preparation method and application thereof

A feline parvovirus and particle technology, applied in the field of genetic engineering, can solve problems such as insufficient HA titer, achieve high safety and effectiveness, increase expression, and high safety

Pending Publication Date: 2022-06-03
CHANGCHUN SR BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the expression of insect baculovirus VP2 has been greatly improved by using the full suspension culture process, but the HA titer is not high enough. At the same time, the purity of VLPs is also one of the important quality indicators of virus-like particle vaccines. The purity of current VLPs Generally around 80%, VLPs with low purity still have some allergic reactions in clinical application, so developing a feline parvovirus-like particle with higher potency and purity is essential for the production of a safer and more effective feline panleukopenia vaccine of great significance and value

Method used

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  • Celine parvovirus-like particle as well as preparation method and application thereof
  • Celine parvovirus-like particle as well as preparation method and application thereof
  • Celine parvovirus-like particle as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Construction of recombinant baculovirus

[0032] The VP2 sequence of the self-isolated FPV JL-125 strain was used to prepare the recombinant baculovirus FPV-VP2 strain. Among them, the FPV JL-125 strain has high homology with the current circulating strain in China. After its VP2 sequence is optimized according to the codons of insect cells, the prepared recombinant baculovirus FPV-VP2 strain can correctly express the FPV VP2 protein. The construction process of the recombinant baculovirus FPV-VP2 strain of the production virus is as follows:

[0033] 1. Optimization and synthesis of VP2 gene

[0034] After sequencing the extracted genome of the FPV JL-125 strain, the VP2 gene sequence was obtained, which was optimized according to the codon preference of Sf9 insect cells. Commonly used codons, adjusted GC content and unfavorable peaks to prolong the half-life of mRNA, those stem-loop structures that affect the stability of mRNA and its binding to ribosomes ...

Embodiment 2

[0040] Example 2 Setting of key preparation process parameters: cell seeding density, rotational speed, harvest time

[0041] 1. The effect of different cell seeding densities on cell growth

[0042] 0.5×10 respectively 6 pcs / ml, 0.8×10 6 pcs / ml, 1.2×10 6 pcs / ml, 1.5×10 6 Cells / ml were inoculated into the bioreactor tank, and the cell density and cell viability were measured daily. The inoculation density was 0.5×10 6 Cells / ml entered the logarithmic growth phase on the 4th day after the preparation period of 3 days, and the cell density reached 9.90×10 on the 8th day. 6 cells / ml, the cell viability was 97.2%; the seeding density was 0.8×10 6 Pieces / ml entered the logarithmic growth phase on the 3rd day, and reached the highest density on the 7th day, 12.1×10 6 cells / ml, the cell viability rate was 95.1%, and then the cells entered the dying stage, and the viable cell density and viability rate decreased; the seeding density was 1.2×10 6 Individuals / ml directly entered ...

Embodiment 3

[0049] Example 3 Preparation of Feline Parvovirus-Like Particles

[0050] Virus Culture: Well-grown Sf9 cells adjusted to 1.2 × 10 6 ~1.5×10 6 The number of cells / ml was inoculated in a bioreactor, and the suspension was fermented and cultured under the conditions of 27°C, 100-110 r / min, and DO 40%-60%. The number of Sf9 cells in the bioreactor reaches 6.0 × 10 6 ~8.0×10 6 When the number of baculoviruses / ml, add an equal volume of fresh medium, inoculate the recombinant baculovirus FPV-VP2 strain constructed in Example 1 at an MOI of 0.1-1.0, at 27°C, 100-110 r / min, DO 40-60% Continue to cultivate under the conditions. The cell cultures were harvested 96-120 hours after inoculation, and the cells were collected and stored frozen at -20°C.

[0051] Cell enrichment and lysis: The harvested cell culture of the recombinant baculovirus FPV-VP2 strain was enriched with a 750KD ultrafiltration hollow fiber column, and 25mmol / LNaHCO with a 4 / 5 ratio of the stock solution was use...

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Abstract

The invention discloses a cat parvovirus-like particle and a preparation method and application thereof.A VP2 sequence of an autonomously separated FPV JL-125 strain is selected for preparing a recombinant baculovirus FPV-VP2 strain, the FPV JL-125 strain and a current Chinese prevalent strain have high homology, after the VP2 sequence is optimized according to insect cell codons, the VP2 sequence is converted into a recombinant baculovirus FPV-VP2 strain, and the recombinant baculovirus FPV-VP2 strain is converted into a recombinant baculovirus FPV-VP2 strain. The prepared recombinant baculovirus FPV-VP2 strain can be used for correctly expressing the FPVVP2 protein. According to the invention, a full suspension culture process is adopted for culture, the expression quantity is greatly improved, and the HA titer can reach 220-221, which is 256-512 times of the culture titer of wild viruses. The expressed protein can be autonomously assembled into complete FPV virus-like particles, the space structure of the protein is similar to that of an original virus, and the virus-like particles are high in titer and higher in safety and have the advantages of stimulating humoral immunity and cellular immunity at the same time. According to the invention, the virus-like particle antigen is prepared by using a genetic engineering means, the novel cat parvovirus-like particle vaccine is prepared, and the vaccine has higher safety and effectiveness.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a cat parvovirus-like particle and a preparation method and application thereof. Background technique [0002] Feline panleukopenia (FP), also known as feline panleukopenia, feline distemper, and feline infectious enteritis, is an acute high-contact infectious disease caused by Feline panleukopenia Virus (FPV). The presentation is characterized by sudden onset of high fever, vomiting, diarrhea, dehydration, and a sharp decrease in the number of circulating leukocytes in the affected cat. FPV mainly infects a variety of animals such as Feline and Mustelidae, especially the incidence and mortality of young animals under 6 months old. The incidence rate of kittens under 1 year old is as high as 83.5%, and the whole litter of kittens will gradually develop the disease. , is the most important infectious disease of felines, posing a serious threat to the life of felines. ...

Claims

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Application Information

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IPC IPC(8): C07K14/015C12N15/866A61K39/23A61P31/14
CPCC07K14/005C12N15/86A61K39/12A61P31/14C12N2750/14323C12N2750/14334C12N2750/14352C12N2800/105C12N2710/14043Y02A50/30
Inventor 夏振强付玉金宏丽王倩郎佳宝方爽爽赵健陈宪平刘瑞雪汪玉彬谷雨洁
Owner CHANGCHUN SR BIOLOGICAL TECH
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