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Novel genetic engineering vaccine of porcine Seneca virus as well as preparation method and application of novel genetic engineering vaccine

A virus and pig plug technology, applied in genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of difficult protein purification, large multiplicity of virus infection and low efficiency.

Active Publication Date: 2019-09-27
苏州世诺生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method of protein purification is difficult, and the efficiency of protein assembly in vitro is very low
Some researchers also used several baculoviruses to express VP1, VP0, and VP3 respectively, and then co-infected Sf9 cells with several viruses to co-express these three proteins, but this method requires a large multiplicity of virus infection, and the same cell needs to be simultaneously Infecting 2 or more viruses, there is a problem of low efficiency
In addition, some researchers use insect cells to express P1, P2, P3 or P1 and 3BC proteins, and expect that by expressing structural proteins and proteases, proteases can digest structural proteins into VP1, VP3 and VP0, but the proteases expressed in this way are toxic , resulting in low yield of structural protein, and low efficiency of protease digestion, resulting in less cleaved VP1, VP3 and VPO proteins

Method used

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  • Novel genetic engineering vaccine of porcine Seneca virus as well as preparation method and application of novel genetic engineering vaccine
  • Novel genetic engineering vaccine of porcine Seneca virus as well as preparation method and application of novel genetic engineering vaccine
  • Novel genetic engineering vaccine of porcine Seneca virus as well as preparation method and application of novel genetic engineering vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0150] Example 1 Construction and Identification of Transfer Vector Dual-VP3-VPO-VP2-VP1-VP4

[0151] 1. Construction and identification of transfer vector Dual-VP3

[0152] 1.1 Amplification and purification of VP3 gene The codon-optimized SVV VP3 gene (SEQ ID NO: 1) was synthesized in Nanjing GenScript and cloned into the pUC17 vector to obtain the pUC-VP3 plasmid vector. The pUC-VP3 plasmid was used as a template, and VP3-F and VP3-R were used as upstream and downstream primers for PCR amplification (the gene sequences of VP3-F and VP3-R are shown in SEQ ID NO.6 and 7). For the amplification system, see Table 1.

[0153] Table 1 VP3 gene amplification system

[0154]

[0155]

[0156] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0157] Perform gel electrophoresis on the PCR product to verify the size of the ...

Embodiment 2

[0244] Example 2 Construction of recombinant baculovirus genome Bac-VP3-VPO-VP2-VP1-VP4

[0245] 1. Transformation of DH10Bac bacteria Take 1 μl of the Dual-VP3-VP0-VP2-VP1-VP4 plasmid in Example 1 and add it to 100 μl of DH10Bac competent cells, mix evenly, ice bath for 30 minutes, heat shock in 42°C water bath for 90 seconds, and then ice bath After 2 minutes, add 900 μl of LB liquid medium without Amp, and incubate at 37° C. for 5 hours. After 100 μl of bacterial solution was diluted 81 times, 100 μl of diluted bacterial solution was spread on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37°C for 48 hours.

[0246] 2. Select single clones and use inoculation needles to pick up large white colonies, then streak on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and culture at 37°C for 48 hours , and then pick a single colony to inoculate the LB liquid medium containing gentamicin, kanamycin, ...

Embodiment 3

[0247] Example 3 Recombinant Baculovirus Transfection

[0248] Inoculate 0.8×10 per well in a six-well plate 6 Sf9 cells, the cell confluence is 50-70%. Prepare the following complexes for each well: dilute 4 μl of Cellfectin transfection reagent with 100 μl of transfection medium T1, briefly vortex; dilute 3 μg of recombinant Bacmid-VP3-VP in Example 2 with 100 μl of transfection medium T1. - VP2-VP1-VP4 plasmids, mix the diluted transfection reagent and plasmids, blow gently to prepare the transfection mixture. Add the above-mentioned transfection complex after the cells adhere to the wall, incubate at 27°C for 5 hours, remove the supernatant, add 2ml of SF-SFM fresh medium, culture at 27°C for 4-5 days, and harvest the supernatant. Obtain the recombinant baculovirus rBac-VP3-VP0-VP2-VP1-VP4, use the MTT relative potency method to detect the virus titer of the harvested P1 generation recombinant baculovirus, rBac-VP3-VP0-VP2-VP1-VP4P1 seed virus The titer is 5.4×10 7 pfu...

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Abstract

The invention discloses an immunological composition which comprises porcine Seneca virus structural protein VP3 and VP1 proteins, as well as porcine Seneca virus structural protein VP2 and / or VP4 protein. Further, the immunological composition can further comprise a porcine Seneca virus structural protein VP0. The immunological composition can be used for preparing a novel genetic engineering subunit vaccine of porcine Seneca virus, the antigenicity, immunogenicity and function of the vaccine are similar to those of natural proteins, the expression level is relatively high, the immunogenicity is strong, and no pathogenicity is caused to animals; the vaccine can be prepared by large-scale serum-free suspension culture in a bioreactor, thereby greatly reducing the cost of vaccine production.

Description

technical field [0001] The invention relates to a genetic engineering vaccine, in particular to a novel genetic engineering vaccine of porcine Seneca virus. The preparation method and application thereof belong to the technical field of animal immune drugs. Background technique [0002] Seneca virus (Seneca Valley Virus, SVV) disease is an animal infectious disease caused by Seneca virus A (SVVA) of the Picornaviridae family. It mainly infects pigs, and pigs of different ages are susceptible. SVV infection causes vesicular lesions in the snout and crown of the hooves, accompanied by clinical manifestations such as lameness, anorexia and lethargy, and the mortality rate of newborn piglets is significantly increased. The infection caused by SVV is indistinguishable from the clinical symptoms caused by foot-and-mouth disease, pig vesicular disease and vesicular stomatitis. Since the disease is a newly discovered zoonotic disease, there is still no commercial vaccine available ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/125A61P31/14C12N15/866C12N15/41
CPCA61K39/125A61K2039/5156A61K2039/552A61P31/14C07K14/005C12N15/86C12N2710/14143C12N2770/32022C12N2770/32034C12N2770/32051C12N2800/105
Inventor 曹文龙孔迪滕小锘易小萍张大鹤
Owner 苏州世诺生物技术有限公司
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