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

A technology of recombinant baculovirus and SARS virus, which is applied in the field of biomedicine, can solve the problems of multiple injections, shortage, and safety problems, and achieve the effects of high application value, increased production, and reduced costs

Active 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] advantage shortcoming therapeutic vaccine The Fc segment of the antibody does not require host recognition Need for antibody humanization inactivated virus vaccine Short time; cheap; relatively easy technology; relatively stable vaccine; no need for refrigeration; easy to transport The production safety conditions are extremely high; there are safety problems; multiple injections are required; the immune response is weak Nucleic 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 vivo Sustained expression of exogenous sources may have some adverse consequences; there are many uncertain factors affecting the immune response induced by nucleic acid vaccines multi-epitope vaccine Can be combined with different types of MHC molecules to achieve efficient presentation and induce strong cellular immunity Lack 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 a high level of humoral immune response

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

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

[0041] 1. Acquisition of gp64-N sequence

[0042] Using the silkworm baculovirus gp64 sequence (SEQ ID NO: 11) as a template, the primers Psp-F, Psp-R, Ptm-F, and 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 N protein gene sequence (SEQ ID NO: 12) of SARS virus was used as a template, and Pn-F and Pn-R were used as primers, and the N protein gene sequence was obtained by PCR amplification. The PCR product was obtained by overlapping PCR to obtain the recombinant target fragment SP-N-TM in which the SP gene sequence, N gene sequence and TM gene sequence were sequentially connected, and the recombinant target fragment was inserted into the p10 promoter of the vector pFastBacDual to construct the recombinant transposable plasmid pFastBacDual- gp64-N uses the polyhedron promoter to promote ...

Embodiment 2

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

[0059] The recombinant transposable plasmid pFastBacDual-gp64-N, 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., operated according to the instructions), and homologous recombination was performed by transposition (the gp64-N sequence on pFastBacDual-gp64-N 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 bacteria fo...

Embodiment 3

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

[0066] The recombinant baculovirus Bmgp64-N was mixed with 3×10 -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 minutes, took 10 μL of the heated mixture for SDS-PAGE analysis, the results showed that the silkworm recombinant baculovirus had expressed N 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 N antigen and a baculovirus displaying an N protein. The recombinant protein SP-N-TM is formed by connecting an N- end of the N 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 N protein is the recombinant baculovirus obtained by the steps of inserting a cording gene of the SP-N-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 an N protein gene of the SARS virus to be fused with a bombyx mori baculovirus envelope protein GP64 gene, realizes display of the N protein on the surface of a viral capsid, and has good immunogenicity and large application value.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a recombinant protein containing the N antigen of SARS virus and a recombinant baculovirus displaying the N 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 842...

Claims

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Application Information

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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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