Recombinant protein containing SARS virus RBD antigen and baculovirus displaying RBD protein

A technology of recombinant baculovirus and SARS virus, applied in the field of biomedicine, can solve the problems of multiple injections, safety problems, shortages, etc., and achieve the effects of high application value, cost reduction, and increase in production

Inactive Publication Date: 2015-01-21
特菲(天津)生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] AdvantagesDisadvantagesTherapeutic vaccineDoes not require Fc fragment of host recognition antibodyneeds Antibody humanizationInactivated virus vaccineShort time; cheap; technology is easier; vaccine is relatively stable; no refrigeration required; easy to transportExtremely high production safety conditions; there are safety problems; multiple injections are required; the immune response is weakNucleic acid vaccine (gene vaccine or DNA vaccine)Simple preparation; easy mass production; good safety, can induce humoral immunity and cellular immune response at the same time; can be continuously expressed in vivoSustained expression of exogenous sources may have some adverse consequences; affect nucleic acid There are many uncertain factors in the vaccine-induced immune response.Multi-epitope vaccinecan be combined with different types of MHC molecules to achieve efficient presentation, and Can induce strong cellular immunityLack of in-depth understanding of SARS-CoV protein epitopes, especially conformational epitopes; synthetic peptides can only cover a small number of linear epitopes, and it is impossible to induce High level of humoral immune response

Method used

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  • Recombinant protein containing SARS virus RBD antigen and baculovirus displaying RBD protein
  • Recombinant protein containing SARS virus RBD antigen and baculovirus displaying RBD protein
  • Recombinant protein containing SARS virus RBD antigen and baculovirus displaying RBD protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of recombinant transposable plasmid pFastBacDual-gp64-RBD

[0041] 1. Acquisition of gp64-RBD sequence

[0042] With the silkworm baculovirus gp64 sequence (SEQ ID NO: 10), the primers Psp-F, Psp-R and Ptm-F, Ptm-R shown in Table 2 were used for PCR amplification to obtain the signal peptide of gp64 (SP ) sequence and transmembrane region (TM) sequence. The RBD protein gene sequence of SARS virus (SEQ ID NO: 11) was used as a template, and the RBD protein gene sequence was obtained by PCR amplification with Prbd-F and Prbd-R as primers. The PCR product was obtained by overlapping PCR to obtain the recombinant target fragment SP-RBD-TM in which the SP gene sequence, RBD gene sequence and TM gene sequence were sequentially connected, and the recombinant target fragment was inserted into the pPolh promoter of the vector pFastBacDual to construct the recombinant transposable plasmid pFastBacDual- gp64-RBD uses the polyhedron promoter pPolh to start ...

Embodiment 2

[0058] Example 2 Obtaining of Bombyx mori recombinant baculovirus Bmgp64-RBD

[0059]The recombinant transposable plasmid pFastBacDual-gp64-RBD, which was successfully identified for recombination, was transformed into Escherichia coli DH10Bac competent cells (purchased from Invitrogen) containing the baculovirus shuttle vector Bacmid, and contained kanamycin, gentamicin, tetracycline, X-gal and IPTG were cultured on LB culture plates (purchased from Shanghai Sangon Biotechnology Co., Ltd., and operated according to the instructions), and homologous recombination was performed by transposition (the gp64-RBD sequence on pFastBacDual-gp64-RBD was transposed by homologous transposition After inserting into the multiple cloning site of Bacmid), the blue and white spots were screened, and the white spots were picked after 48 hours of dark culture, and the white spots were kept in the LB culture medium containing tetracycline, kanamycin, gentamycin, X-gal and IPTG After shaking the ...

Embodiment 3

[0065] Example 3 Expression of RBD protein in silkworm BmN cells

[0066] The recombinant baculovirus Bmgp64-RBD was 6×10 -6 A dose of pfu / cell was used to infect silkworm BmN cells for virus amplification. After 3 to 5 days of infection, the virus liquid was collected, separated and purified, and 10 μL of the supernatant was added to an equal volume of 2× protein loading buffer (100Mm Tris-HCl, 4% SDS, 0.15% bromophenol blue, 10% glycerol), heated at 100°C for 10 min, took 10 μL of the heated mixture for SDS-PAGE analysis, the results showed that the silkworm recombinant baculovirus had expressed RBD protein, protein sequencing results Shown, its amino acid sequence is shown in SEQ ID NO:1.

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Abstract

The invention discloses a recombinant protein containing an SARS virus RBD antigen and a baculovirus displaying an RBD protein. The recombinant protein SP-RBD-TM is formed by connecting an N- end of the RBD protein of an SARS virus with a signal peptide SP of a baculovirus envelope protein GP64, and connecting a C- end with a transmembrane domain TM of the baculovirus envelope protein GP64. The recombinant baculovirus having the surface displaying the SARS antigen RBD protein is the recombinant baculovirus obtained by the steps of inserting a cording gene of the SP-RBD-TM into a donor plasmid, carrying out homologous recombination with a genome of a shuttle vector Bacmid through transposition to obtain a recombinant baculovirus genome, then transfecting a bombyx mori cell with the recombinant baculovirus genome, and packaging in the cell to obtain the recombinant baculovirus. The recombinant baculovirus allows the RBD protein of the SARS virus to be fused with the bombyx mori baculovirus envelope protein GP64, realizes display of the RBD protein on the surface of a viral capsid, and has good immunogenicity.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a recombinant protein containing a SARS virus RBD antigen and a recombinant baculovirus displaying the RBD protein. Background technique [0002] Severe Acute Respiratory Syndrome (Severe Acute Respiratory Syndromes), also known as infectious atypical pneumonia, referred to as SARS, is a new respiratory infectious disease caused by infection with the SARS coronavirus. It is mainly transmitted through close-range air droplets, with fever, headache, muscle aches, fatigue, dry cough and less sputum as the main clinical manifestations, and respiratory distress may occur in severe cases. The disease is highly contagious, and there are significant clusters in families and hospitals. The first case, also the first in the world, appeared in Foshan, Guangdong in November 2002, and quickly became an epidemic. From November 2002 to August 5, 2003, 29 countries reported a total of 8422...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/866C12N15/66A61K48/00A61P31/14
Inventor 张耀洲闫晶晶舒特俊陈剑清盖其静
Owner 特菲(天津)生物医药科技有限公司
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