Arg-gly-asp (RGD)-fused porcine circovirus 2 (PCV2) virus-like particles (VLPs), mutant infectious clone and preparation method of RGD-fused PCV2 VLPs, and application of RGD-fused PCV2 VLPs and infectious clone

A technology for porcine circovirus and type 2 virus, applied in the field of molecular biology and virology, can solve the problems of poor reduction ratio of viral load in pigs, low antibody titer, long immune blank period, etc., to enhance humoral immune response , the effect of increased antibody levels

Active Publication Date: 2020-01-10
湖南派智生物科技有限公司
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] The invention provides a porcine circovirus type 2 virus-like particle fused with RGD, an infectious clone and its preparation method and application, which solves the

Method used

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  • Arg-gly-asp (RGD)-fused porcine circovirus 2 (PCV2) virus-like particles (VLPs), mutant infectious clone and preparation method of RGD-fused PCV2 VLPs, and application of RGD-fused PCV2 VLPs and infectious clone
  • Arg-gly-asp (RGD)-fused porcine circovirus 2 (PCV2) virus-like particles (VLPs), mutant infectious clone and preparation method of RGD-fused PCV2 VLPs, and application of RGD-fused PCV2 VLPs and infectious clone
  • Arg-gly-asp (RGD)-fused porcine circovirus 2 (PCV2) virus-like particles (VLPs), mutant infectious clone and preparation method of RGD-fused PCV2 VLPs, and application of RGD-fused PCV2 VLPs and infectious clone

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preparation example Construction

[0051] According to another aspect of the present invention, there is also provided a method for preparing porcine circovirus type 2 (PCV2) virus-like particles fused with RGD, comprising the steps of: designing primers using Oligo7.0 for mutants in the Loop CD region ; Using pET100-PCV2-DNLS Cap as a template, use over-lap PCR technology and gel recovery to amplify the target fragment of the mutant; insert the target fragment into the vector to obtain the recombinant expression plasmid pET100-PCV2 Cap-iRGD; the recombinant expression plasmid The recombinant genetically engineered bacteria are obtained by transforming into competent cells; the recombinant genetically engineered bacteria are separated and purified to obtain recombinant proteins; the recombinant proteins are assembled in vitro to obtain porcine circovirus type 2 (PCV2) virus-like particles fused with RGD. The present invention finds that when the RGD short peptide is inserted into the N-terminus of the Loop CD re...

Embodiment 1

[0070] 1.1 Construction of recombinant plasmid with RGD inserted into PCV2 Cap protein and 3D protein structure simulation of mutants

[0071] The pET100-PCV2-DNLS Cap plasmid containing the PCV2 ORF2 protein sequence (GenBank: JF504708) preserved in the laboratory was used as a template, and the amino acid sequence of pET100-PCV2-DNLS Cap is shown in SEQ ID NO: 13, and the experimental design was carried out. Use DNAMAN software to find the 75-92 amino acids (NINDFLPPGGGSNPRSVP) of the Loop CD region of PCV2 Cap. It is known that when PCV2 Cap is assembled into VLPs in vitro, the LPPGGGSN motif is located on the outer surface of VLPs. Mutations are carried out on these 8 amino acids. 85 G and 86 The insertion between S contains a copy of RGD sequence, named pET100-PCV2 Cap-iRGD.

[0072] Using the known PCV2cs protein structure (Protein Data Bank number: 3R0R), through Swiss-Model protein structure simulation software (http: / / swissmodel.expasy.org / ) and Modeller online homol...

Embodiment 2

[0123] IFA analysis of cell invasion characteristics of PCV2 virus-like particles fused with RGD

[0124] PK15 and IPEC-J2 cells were inoculated on culture plates filled with cell slides, and 1ug of WT-VLPs and iRGD-PCV2 VLPs were added, respectively. Untreated cells were used as controls. After incubation for 1 hour, fresh medium was replaced and incubated overnight. The cells were fixed with 4% paraformaldehyde for 20 min at room temperature, and the cells were permeabilized with 0.1% Triton for 10 min at room temperature. After blocking with 3% BSA for 1 hour, rabbit-derived PCV2 polyclonal antibody (1:500) and FITC-labeled donkey anti-rabbit secondary antibody (1:2000) were diluted with 1% BSA, and incubated at 37°C for 1 hour. After washing three times with PBST (containing 0.05% Tween), the nuclei were stained with DAPI, and the slides were sealed with nail polish and observed under an inverted fluorescent microscope.

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Abstract

The invention discloses arg-gly-asp (RGD)-fused porcine circovirus 2 (PCV2) virus-like particles (VLPs), an infectious clone and a preparation method of the RGD-fused PCV2 VLPs, and application of theRGD-fused PCV2 VLPs and the infectious clone. According to the RGD-fused PCV2 VLPs, RGD oligopeptides are fused on PCV2 Cap proteins. According to the RGD-fused PCV2 VLPs, the PCV2 Cap proteins are subjected to RGD polypeptide sequence oligopeptide modification, the RGD oligopeptides are fused on the PCV2 Cap proteins, and the PCV2 VLPs with RGD-fused on the surfaces are assembled in vitro. Compared with wild PCV2 VLPs, the levels of PCV2 specific IgM and IgG antibodies generated by RGD-fused PCV2 VLP immune mice are significantly increased, high-titer PCV2 antibodies can be generated in an induced mode, and the RGD-fused PCV2 VLPs enhance humoral immunity response. The RGD-fused PCV2 VLPs can be applied to development of enhanced VLPs and differential diagnosis molecular label VLPs vaccines, and a new idea is provided for research and development of PCV2 vaccines.

Description

technical field [0001] The invention relates to the fields of molecular biology and virology, in particular to a porcine circovirus type 2 (PCV2) virus-like particle and mutant infectious clone fused with RGD. In addition, the present invention also relates to a preparation method and application of porcine circovirus type 2 (PCV2) virus-like particles and mutant infectious clones comprising the fusion RGD. Background technique [0002] Porcine circovirus (PCV for short) belongs to Circoviridae, is a kind of non-encapsulated, icosahedral single-stranded circular DNA virus, with a genome size of about 1.7Kb. There are currently three genotypes of PCV, namely porcine circovirus 1 (PCV1 for short), porcine circovirus 2 (PCV2 for short) and the newly discovered porcine circovirus type 3 (Porcine circovirus 1). circovirus 3, referred to as PCV3). According to relevant reports, PCV1 is not pathogenic, while PCV2 can cause many related diseases, and whether PCV3 is pathogenic is ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N7/01C12N15/70A61K39/12A61P31/20C12R1/93
CPCA61K39/12A61P31/20C07K5/0817C07K14/005C07K2319/00C12N7/00C12N15/70C12N2750/10021C12N2750/10023C12N2750/10034C12N2750/10051
Inventor 王乃东蒋一凡王东亮湛洋李周勉袁晓民
Owner 湖南派智生物科技有限公司
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