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Chimeric virus-like particle vaccine and preparation method therefor and application of chimeric virus-like particle vaccine

A technology of chimeric viruses and particles, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as costing a lot of manpower and material resources, and achieve improved immunogenicity, immunogenicity retention, and clear ingredients Effect

Active Publication Date: 2020-09-11
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to ensure the effectiveness of the vaccine, vaccine manufacturers need to change the vaccine strain according to the prevalence of wild strains, which requires a lot of manpower and material resources

Method used

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  • Chimeric virus-like particle vaccine and preparation method therefor and application of chimeric virus-like particle vaccine
  • Chimeric virus-like particle vaccine and preparation method therefor and application of chimeric virus-like particle vaccine
  • Chimeric virus-like particle vaccine and preparation method therefor and application of chimeric virus-like particle vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of porcine Seneca Valley virus, circovirus type 2 chimeric plasmid

[0037] (1) Cloning of P1 Gene of Porcine Seneca Valley Virus

[0038]Primers were designed according to the published porcine Seneca Valley virus gene sequence (Genbank: KY747510.1):

[0039] Primer1: 5'-GGTAATGTTCAGACAACC-3',

[0040] Primer2: 5'-CTGTTCCTCGTCCGTCCC-3',

[0041] Primer3: 5'-TCCACCGACAACGCCGAG-3',

[0042] Primer4: 5'-TTGCATCAGCATCTTCTG-3',

[0043] Primer5: 5'-GGGCCTATTCCCACAGCA-3',

[0044] Primer6: 5'-GTGGAACACGTAGGAAGG-3'.

[0045] Extract the total RNA of porcine Seneca Valley virus as a template, synthesize the first strand of cDNA by MLV-reverse transcriptase, and use Primer1 and Primer 2 as primers to amplify the VP0 fragment by PCR. The PCR reaction system is 50 μL, and the PCR reaction conditions are: Pre-denaturation at 95°C for 5min, 30sec at 95°C, 30sec at 60°C, 1min30sec at 72°C, 30cycles, 10min at 72°C, 10min at 16°C.

[0046] The PCR product...

Embodiment 2

[0058] Example 2 Construction and extraction of rFB-P1-PCV2 recombinant bacmid

[0059] The rFB-P1-PCV2 recombinant bacmid was transformed into DH10Bac competent cells, and after 4 hours at 37°C, it was coated with kanamycin (50 μl / mL), tetracycline (10 μl / mL), gentamicin (7 μg / mL), X-Gal (100 μl / mL), IPTG (40 μl / mL) on the LB blue and white spot screening plate, after culturing, pick white colonies for PCR identification, obtain rFB-P1-PCV2 recombinant vector, expand the culture, The recombinant bacmid was extracted by isopropanol precipitation and used immediately or stored at -20°C for later use.

Embodiment 3

[0060] Example 3 The acquisition and identification of chimeric recombinant baculovirus

[0061] (1) Obtain chimeric recombinant baculovirus

[0062] Transfect sf9 cells with rFB-P1-PCV2 by liposome transfection method, place them for culture at 28°C, and after 48-72 hours, the cells become round and large, and a large number of granules appear inside, and then the cells die in large numbers; cells in the negative control group Normal growth indicated that the recombinant baculovirus was assembled successfully. Harvest the culture, centrifuge at 1500g for 5-10min to collect the supernatant, which is the F1 generation virus solution; then use the F1 generation virus solution to infect the blank sf9 cells, and harvest the culture supernatant when the resulting cytopathic changes reach more than 80%, which is the F2 generation Substitute virus fluid. According to this method, the recombinant baculoviruses are transferred to the F4 generation, and all harvested recombinant bacul...

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Abstract

The invention relates to a chimeric virus-like particle vaccine and a preparation method therefor and application of the chimeric virus-like particle vaccine, in particular to a porcine Seneca valleyvirus and porcine circovirus-2 chimeric virus-like particle vaccine. According to the chimeric virus-like particle vaccine, sequences of VP0, VP3 and VP1 are connected in tandem for expression, wherein part of the VP3 sequences are replaced with porcine circovirus-2 ORF2 gene C-terminal epitope sequences to form a recombination sequence; the recombination sequence transfects sf9 cells after beingconstructed on recombinant bacmid for further expression to obtain porcine Seneca valley virus and porcine circovirus-2 chimeric virus-like particles. It is the first time for the invention to developthe porcine Seneca valley virus and porcine circovirus-2 chimeric virus-like particle vaccine by utilizing the chimeric virus-like particles, and the porcine Seneca valley virus and porcine circovirus-2 chimeric virus-like particle vaccine can exert a relatively good immune protection effect on the porcine Seneca valley virus and the porcine circovirus-2; and the vaccine of the invention is economical and practical, can effectively reduce the epidemic prevention cost of diseases, and provides a new method of preventing two diseases at the same time for breeding enterprises in China.

Description

technical field [0001] The invention relates to the field of veterinary biological products, in particular to a chimeric virus-like particle vaccine and its preparation method and application. Background technique [0002] Seneca Valley virus (SVV), also known as Seneca virus type A, is a single-stranded positive-sense RNA virus and the only member of the genus Senecavirus in the Picornaviridae family. Pigs of different ages and different breeds are susceptible, and the incidence rate of sows is high, up to 70%-90%, but the mortality rate of infected pigs is very low. For newborn piglets, the morbidity rate can reach 70%, and the mortality rate is between 15%-30%. Therefore, SVV poses a greater threat to the production and economic benefits of the pig industry. Vaccination is considered to be an effective means to prevent and control the epidemic of Seneca Valley virus, and there is currently no commercial SVV vaccine on the market. [0003] The baculovirus expression sys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/866A61K39/125A61K39/12A61K39/39A61P31/14A61P31/20
CPCC07K14/005C12N15/86A61K39/12A61K39/39A61P31/14A61P31/20C12N2770/32022C12N2770/32023C12N2770/32034C12N2770/32051C12N2750/10022C12N2750/10023C12N2750/10034C12N2750/10051C07K2319/00C12N2710/14043A61K2039/5258A61K2039/552A61K2039/70
Inventor 何至远张贺楠吴珊珊王贵华肖进
Owner CHINA ANIMAL HUSBANDRY IND
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