Novel genetically-engineered subunit vaccine to foot-and-mouth disease virus O

A technology of foot-and-mouth disease virus and coding gene, which is applied in the field of O-type foot-and-mouth disease virus new genetic engineering subunit vaccine, can solve the problems of low assembly efficiency, large multiplicity of virus infection, and low efficiency, and achieve reduced production costs, strong immunogenicity, high level of expression

Active Publication Date: 2019-09-20
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

[0007] At present, some studies use Escherichia coli to express the three proteins of VP1, VP3 and VP0 respectively, and then assemble them in vitro. This method is difficult for protein purification, and the efficiency of protein assembly in vitro is very low; other studies use multiple baculoviruses to express them separately. VP1, VP0 and VP3 proteins, and then co-infect Sf9 cells with multiple viruses to co-express these three proteins. This method requires a large multiplicity of virus infection, and the same cell needs to be infected with 2 or more viruses at the same time, and the efficiency is very high. low problem

Method used

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  • Novel genetically-engineered subunit vaccine to foot-and-mouth disease virus O
  • Novel genetically-engineered subunit vaccine to foot-and-mouth disease virus O
  • Novel genetically-engineered subunit vaccine to foot-and-mouth disease virus O

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 Embodiment 1

[0138] Example 1 Example 1 Construction and Identification of Transfer Vector Dual-VP3-VPO-VP2-VP1-VP4

[0139] 1. Construction and identification of transfer vector Dual-VP3

[0140] 1.1 Amplification and purification of the VP3 gene The codon-optimized 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.2 and 3), and the amplification system is shown in Table 1.

[0141] Table 1 VP3 gene amplification system

[0142]

[0143]

[0144] 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.

[0145] Perform gel electrophoresis on the PCR product to verify...

Embodiment 2

[0227] Example 2 Construction of recombinant baculovirus genome Bac-VP3-VPO-VP2-VP1-VP4

[0228] 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.

[0229] 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

[0230] Example 3 Recombinant Baculovirus Transfection

[0231] 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 immunization composition, comprising foot-and-mouth disease virus O structural proteins VP3 and VP1, and foot-and-mouth disease virus O structural proteins VP2 and / or VP4. Further, the immunization composition also comprises a foot-and-mouth disease virus O structural protein VP0. The immunization composition is applicable to the preparation of a novel genetically-engineered subunit vaccine to foot-and-mouth disease virus O; the novel genetically-engineered subunit vaccine is similar to natural proteins in terms of antigenicity, immunogenicity and functionality, the expression level is high, the immunogenicity is high, and the novel genetically-engineered subunit vaccine is non-pathogenic to an animal. In addition, the novel genetically-engineered subunit vaccine is suitable for large-scale serum-free suspension culture through biosensors; vaccine production cost is reduced greatly.

Description

technical field [0001] The invention relates to the technical field of animal immune medicines, in particular to a novel genetic engineering subunit vaccine of O-type foot-and-mouth disease virus. Background technique [0002] Foot-and-mouth disease (foot-and-mouth disease, FMD) is a disease produced by foot-and-mouth disease virus (foot and mouthdisease virus, FMDV) infecting artiodactyls (such as pigs, cattle, sheep, deer, etc.), which is characterized by Infected artiodactyls will develop blisters on the mouth, feet and other parts of the skin, resulting in the death of some animals. [0003] FMDV belongs to the genus Aphthovirus of the Picornaviridae family and is the causative agent of a highly contagious disease (foot-and-mouth disease) in artiodactyls. In the center of the virus is a single-stranded positive-strand RNA, consisting of about 8,000 bases, which is the basis of infection and heredity; surrounded by proteins that determine the antigenicity, immunity and s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/09C12N15/866C12N5/10A61K39/135A61P31/14
CPCA61K39/12A61K2039/552A61P31/14C07K14/005C12N15/86C12N2710/14043C12N2770/32122C12N2770/32134
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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