Method for producing recombinant vaccine by using plant membrane system

A recombinant vaccine and membrane system technology, applied in the field of recombinant vaccine production using plant membrane systems, can solve the problems of further improvement of the immunogenicity of recombinant vaccines, and achieve easy storage and distribution, good genetic stability, and high immunogenicity Effect

Active Publication Date: 2022-03-01
猎境(嘉兴)生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the deficiencies in the prior art, the present invention provides a method for producing a recombinant vaccine using a plant membrane system, which solves the problem of recombination in the prior art Immunogenicity of vaccines needs to be further improved

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  • Method for producing recombinant vaccine by using plant membrane system
  • Method for producing recombinant vaccine by using plant membrane system
  • Method for producing recombinant vaccine by using plant membrane system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of porcine parvovirus surface antigen and chloroplast outer membrane protein fusion vector and Agrobacterium transformation

[0045] 1. Carrier construction

[0046] Synthesize the gene sequence PPV-TOC34 (SEQ ID NO.8) fused with the surface antigen of porcine parvovirus and the transmembrane region of chlorophyll outer membrane protein TOC34, and connect it to the pYBA1132 plant expression vector by double enzyme digestion to obtain the recombinant plasmid DNA (1132-PPV- TOC34). After the ligation product was transformed into Escherichia coli DH5α competent cells, positive clones were obtained by kanamycin screening. Bacterial liquid PCR verified the insertion of PPV-TOC34, and sequenced to further verify the correctness of the sequence. The expression vector map of 1132-PPV-TOC34 is as follows: figure 1 shown.

[0047] 2. Agrobacterium transformation

[0048] Take Agrobacterium GV3101 competent cells (100 μL) stored at -80°C and thaw on ice....

Embodiment 2

[0049] Example 2 Fusion expression of porcine parvovirus surface antigen and chloroplast outer membrane protein

[0050] 1. Preparation of Agrobacterium

[0051] Resuscitate the constructed frozen Agrobacterium, take 5 mL of LB medium with a final concentration of 50 mg / L kanamycin in a 10 mL centrifuge tube, and insert 1% of the inoculated amount, that is, 50 μL of the bacterial solution into the LB medium , 28°C, 200rpm in a shaker for overnight shaking culture. Then expand the culture, take 20 mL of LB medium with a final concentration of 50 mg / L kanamycin in a 50 mL centrifuge tube, insert 1% of the inoculum, that is, 200 μL of the bacterial solution into the LB medium, and set the temperature at 28 ° C and 200 rpm. Cultivate on a shaking table until the OD value of Agrobacterium grows to about 0.5-1.0. Add 20 μL of 100 mmol / L acetosyringone, and continue to shake and incubate for 2 hours.

[0052] 2. Leaf disc transformation

[0053] Select the first fully expanded he...

Embodiment 3

[0054] Example 3 Verification and activity determination of fusion expression of porcine parvovirus surface antigen and chloroplast outer membrane protein

[0055] 1. Immunohistochemistry

[0056] Tobacco leaves are cut into thin shreds, put into the enzymolysis solution (1.5% cellulase, 0.4% pectinase, 0.4M mannitol, 20mM KCl, 20mM MES, 10mM CaCl 2 ) in a petri dish in the dark at 70rpm overnight. The protoplasts were filtered, centrifuged at 60 g, and the supernatant was discarded after centrifugation to retain the precipitate. The pellet was resuspended with 5 mL of WA buffer (Dongsheng Biological) solution. After suspension, use 60g, centrifuge for 10min, discard the supernatant, and keep the precipitate. Add 1 mL of WA buffer solution containing 4% paraformaldehyde to the precipitation, and incubate at room temperature at 60 rpm for 1 h. After immobilization, mouse FITC-labeled anti-PPV monoclonal antibody (VMRD, USA) was incubated. After washing with TBST solution, ...

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Abstract

The invention provides a method for producing a recombinant vaccine by using a plant membrane system. Comprising recombinant DNA for expressing and coding a recombinant vaccine, and the recombinant vaccine comprises a recombinant vaccine protein and a fusion protein formed by anchoring polypeptide positioned on a plant membrane. The invention solves the problem of low immunogenicity in the prior art. The fusion protein formed by the recombinant vaccine protein and the anchoring polypeptide is adopted, so that the immunogenicity of the recombinant vaccine is obviously improved.

Description

technical field [0001] The invention relates to the technical field of recombinant DNA, in particular to a method for producing a recombinant vaccine using a plant membrane system. Background technique [0002] At present, most of the recombinant protein drugs in clinical use come from mammalian cells and Escherichia coli expression and production platforms, and a small part come from yeast and insect cells. Similar to mammalian cells, plant cells also have an inner membrane system, so they can use molecular chaperones to fold and assemble recombinant proteins. Compared with animal expression systems, plant expression systems lack animal pathogenic pollutants and the culture medium is cheap (Schillberg et al., Journal of plant physiology 258(2021): 153359.). [0003] The plant production platform used as a bioreactor to produce exogenous proteins can be transgenic plants or suspension culture cells, which can be expressed stably or transiently. The stable expression system...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/23A61P31/20
CPCC12N9/16C12N9/12C12N9/1051C07K14/415C12Y301/05001C12Y207/11017A61K39/12A61P31/20C07K2319/03C07K2319/09A61K2039/517A61K2039/575
Inventor 缪国鹏相深杨纬经
Owner 猎境(嘉兴)生物科技有限公司
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