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Design of broad-spectrum rabies virus-like particle antigen and stable expression cell strain HEK-293 thereof

A technology of HEK-293 and rabies virus, which is applied in the field of vaccines and can solve the problems of carrier virus contamination and other issues

Pending Publication Date: 2022-04-01
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, such vaccines may be contaminated with infectious carrier viruses

Method used

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  • Design of broad-spectrum rabies virus-like particle antigen and stable expression cell strain HEK-293 thereof
  • Design of broad-spectrum rabies virus-like particle antigen and stable expression cell strain HEK-293 thereof
  • Design of broad-spectrum rabies virus-like particle antigen and stable expression cell strain HEK-293 thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Broad-spectrum rabies virus-like particle (RVLPs) antigen design

[0070] 1. Sequence Analysis of RVGP

[0071] The nucleotide sequences of CSV strain RVGP and RVMP were obtained from NCBI database, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The sequence analysis and alignment results of the candidate RVGP are as follows: figure 1 shown.

[0072] The B cell epitopes (Table 1), CLT cell epitopes and Th cell epitopes (Table 2) of RVGP measured by bioinformatics software are all located in the extracellular domain of mature RVGP.

[0073] Table 1 B cell epitope analysis of CSV strain RVGP

[0074]

[0075] Table 2 T cell epitope analysis of CSV strain RVGP

[0076]

[0077]

[0078] 2. Prediction of RVGP glycosylation sites

[0079] The mature RVGP ectodomain has three potential N-glycosylation sites (Asn37, Asn247 and Asn319), namely the Asn56, Asn266 and Asn338 sites of immature RVGP. N-glycosylation affects the antigenicity and corre...

Embodiment 2

[0085] Example 2 Construction of recombinant plasmid pcDNA3.1(+)-RVLPs-EGFP

[0086] The whole gene synthesis of RVGP and RVMP was constructed into pcDNA3.1(+). Use pcDNA3.1(+) as template to amplify CMV, SV40 PA and EGFP respectively; then use CMV and SV40 PA as templates to amplify target fragments (G) SV40PA-CMV and (M) SV40 PA-CMV by overlap extension PCR ; Finally, using (G)SV40 PA-CMV and EGFP as templates, the target fragment (G)SV40 PA-CMV-EGFP was amplified by overlap extension PCR.

[0087] The electrophoresis results of the obtained target fragments are as follows: Figure 6 As shown, it is consistent with the actual size (CMV:618bp; SV40 PA:122bp; (G) / (M)SV40 PA-CMV:760bp; EGFP:759bp; (G)SV40 PA-CMV-EGFP:1484bp), indicating the construction success.

Embodiment 3

[0088] Example 3 Construction of HEK-293 eukaryotic expression system

[0089] 1. Construction process of eukaryotic expression system

[0090] Figure 7 The construction of pcDNA3.1(+)-RVLPs-EGFP and the procedure for obtaining the HEK-293 cell line stably expressing RVLPs are shown, as follows:

[0091] 1) The eukaryotic plasmid SV40 PA-CMV-EGFP was transfected into HEK-293 cells, and the cells were cultivated for cell line selection of stably expressing RVLPs;

[0092] 2) 48h after transfection, the protein and mRNA levels of RVLPs were detected by WB and qPCR;

[0093] 3) Use 2 mg / mL G418 to screen HEK-293 cell lines stably expressing broad-spectrum rabies virus-like particle antigens, and after culture and enrichment, the positive clones are amplified and passaged;

[0094] 4) Digest the positive monoclonal cell line, use flow cytometry to detect the proportion and average fluorescence intensity of EGFP positive cells, and use WB to detect the expression of RVLPs, and ...

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Abstract

The preparation method comprises the following steps: firstly, designing a broad-spectrum rabies virus-like particle antigen by using RVLPs, which is self-assembled by glycoprotein RVGP and matrix protein RVMP of a CVS strain rabies virus RABV, as an antigen; then, the RVGP, the RVMP and the EGFP are jointly constructed into a eukaryotic expression vector pcDNA3.1 (+), and the eukaryotic expression vector is named as pcDNA3.1 (+)-RVLPs-EGFP. Each protein has an independent promoter (CMV), a ribosome binding site (Kozak sequence) and a PloyA tail (PA) so as to ensure the surface level of each protein, and meanwhile, EGFP (enhanced green fluorescent protein) is used for monitoring the expression of the target protein in real time so as to construct a eukaryotic recombinant expression plasmid; and then transfecting HEK-293 cells with the eukaryotic recombinant expression plasmids, carrying out amplification and passage on the cells, screening and identifying, and screening to obtain the positive monoclonal cell strain HEK-293 / RVLPs capable of stably and efficiently expressing RVLPs. The obtained antigen has post-translational modification of mammalian cells, the defect that RVLPs derived from bacteria, yeast, insects and plant cells show low immunogenicity due to incorrect glycosylation modification is overcome, and a foundation is laid for large-scale production of the RVLPs antigen.

Description

technical field [0001] The invention belongs to the technical field of vaccines, and in particular relates to the design of a broad-spectrum rabies virus-like particle (RVLPs) antigen and the screening and identification of its stable expression cell line HEK-293. Background technique [0002] Rabies (Rabies) is an ancient and most neglected zoonotic disease, mainly caused by the lethal pathogen-rabies virus (RABV) (Zhao R Q, Shan Y, Li M H, et al. NovelStrategy for Expression and Characterization of Rabies Virus Glycoprotein[J].Protein Expression and Purification,2020,168:105567.Liu,Zhao W,He W T,etal.Generation of Monoclonal Antibodies against Variable Epitopes of the MProtein of Rabies Virus[J]. Viruses, 2019, 11(4).). Dogs are the main host of RABV, and more than 99% of human rabies are mediated by dogs (Navid M T, Li YY, Zhou M, et al.Comparison of the Immunogenicity of Two Inactivated Recombinant Rabies Viruses Overexpressing the Glycoprotein[J].Archives of Virology,...

Claims

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Application Information

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IPC IPC(8): C07K14/145C12N15/85C12N15/47C12N15/65C12N5/10A61K39/205A61P31/14
Inventor 马兴元赵章婷郑文云邓昌平
Owner EAST CHINA UNIV OF SCI & TECH
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