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Fusion protein of rabies virus G protein expressing Fc fragment and preparation method thereof

A rabies virus and G protein technology, applied in the field of genetic engineering, can solve the problems of lack of immunization methods and insufficient rabies immunity, etc., and achieve the effect of broad application prospects

Active Publication Date: 2019-08-06
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows scientists to create stable raby virus particles that are able to carry different types of proteins like gamma glovesan monkey disease virus type 1 envelope genetic material from their wildtype strain into humans through an infected skin wound caused by rabid dog bitten during pregnancy. These tiny objects called rbcagles were found to be highly effective at delivering these protectants against raborid rodents without causing harmful side reactions on animals they come naturally close enough together to take them back down again.

Problems solved by technology

This patented technical problem addressed in this patents relates to developing better ways to protect children who may be exposed during epidemics due to rabid roditis viruses such as Lassa fevers, while avoiding injectable antibodies like ruffles tickets used in traditional methods.

Method used

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  • Fusion protein of rabies virus G protein expressing Fc fragment and preparation method thereof
  • Fusion protein of rabies virus G protein expressing Fc fragment and preparation method thereof
  • Fusion protein of rabies virus G protein expressing Fc fragment and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Construction of SHG-Fc Fusion Protein SHG-Fc Recombinant Expression Vector

[0072] 1. Design of DNA sequence of SHG-Fc fusion protein

[0073] Link the 3' end of the extracellular region of the G protein of the rabies virus GD-SH strain with a Linker (amino acid sequence: GSGGGGSGGGGSGS) to the IgG2a Fc fragment (the hinge region and the CH2 and CH3 of the constant region), wherein the Fc sequence is located in the rabies The carboxyl terminal of the viral G protein sequence, and the complement site of the mutated Fc fragment, the IgG2a Fc fragment 318th Glu, 320th Lys, and 322th Lys are replaced by ALa to obtain the coding gene of the SHG-Fc fusion protein The nucleotide sequence of the fusion protein is shown in SEQ ID NO: 1, and the amino acid sequence of the corresponding fusion protein is shown in SEQ ID NO: 2.

[0074] Further, the baculovirus GP64 signal peptide and the mouse Kappa chain leader sequence were inserted at the 5' end, figure 1 It is a s...

Embodiment 2

[0104] Example 2 Transformation of recombinant plasmids into DH10Bac Escherichia coli to obtain recombinant shuttle vector Bacmid

[0105] 1. Experimental method

[0106] 1. Transform the recombinant plasmid into Escherichia coli DH10Bac competent bacteria

[0107] Transform DH10Bac competent cells with the recombinant plasmid SHG-Fc-PfastBacDuaL according to the instructions of the Bac-to-Bac expression system. Homologous recombination occurred to obtain the recombinant Bacmid plasmid rBac-SHG-Fc containing the SHG-Fc gene, which realized the transfer of foreign genes into the baculovirus backbone vector, and finally obtained 10 -1 、10 -2 Two gradient coated panels.

[0108] 2. Selection of recombinant bacteria and plasmid extraction

[0109] After 48 hours of cultivation, pick the large white colonies on the plate for the second blue-white screening, pick the colonies that are still white after the second screening, and inoculate 3.0 mL containing tetracycline (10 μg / mL) a...

Embodiment 3

[0118] Embodiment 3 Acquisition of recombinant baculovirus

[0119] 1. Experimental method

[0120] 1. Transfection of recombinant plasmids into Sf9 cells

[0121] Transfection was performed on a 6-well plate, and the cells were in the logarithmic phase (1.5-2.5×10 6 cells / mL), and the survival rate was higher than 95%, according to the transfection reagent CeLLfectin TM II Reagent (Thermo Fisher) instruction manual step operation, set up wild-type Bacmid transfection control at the same time for transfection, in order to obtain wild-type baculovirus. After the transfection, the cells were placed in the incubator, and the cytopathic changes were observed every day.

[0122] 2. Acquisition and propagation of recombinant virus

[0123] After transfection, observe the cell pathological changes every day, continue to cultivate until the 5th day, harvest the culture supernatant, 2000×g, centrifuge for 10min, transfer the supernatant to a new sterilized centrifuge tube, this is...

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Abstract

The invention discloses a fusion protein of a rabies virus G protein expressing an Fc fragment and a preparation method thereof. A gene encoding the rabies virus G protein fusing and expressing the Fcfragment has the nucleotide sequence shown in SEQ ID NO:1, and the rabies virus G protein fusing and expressing the Fc fragment has the amino acid sequence shown in SEQ ID NO:2. The rabies virus G protein and the Fc fragment are fused and expressed for the first time. Recombinant baculovirus constructed by a baculovirus expression system has stable expression of foreign proteins and stable potency. Due to fusion with the Fc fragment, the fusion protein can cross a mucosal barrier, is transported to a lamina propria, is ingested by antigen-presenting cells, is presented to T cells to induce adaptive immunity of a body, a new strategy is provided for research and preparation of oral rabies vaccine and broad research prospects are achieved in prevention and treatment of rabies.

Description

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Claims

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Application Information

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Owner SOUTH CHINA AGRI UNIV
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