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Respiratory syncytial virus virus-like particle vaccine as well as preparation method and application thereof

A technology for syncytial virus and respiratory tract, which is applied in the field of immune prevention of respiratory syncytial virus infection in humans and susceptible animals, and can solve problems such as the imbalance of cytokine ratio

Inactive Publication Date: 2015-01-21
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The inability of FIV vaccination to induce an effective antiviral immune response and cause VED is a major obstacle in the development of current RSV vaccines
The reason is that FIV vaccination cannot induce the body to produce high levels of neutralizing antibodies, and fails to induce antiviral effects CD8 + T cell immune response, host Th1 / Th2 ratio and imbalance of Th1 and Th2 cytokines, etc.

Method used

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  • Respiratory syncytial virus virus-like particle vaccine as well as preparation method and application thereof
  • Respiratory syncytial virus virus-like particle vaccine as well as preparation method and application thereof
  • Respiratory syncytial virus virus-like particle vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] [Example 1] Construction and detection of respiratory syncytial virus virus-like particles (VLPs)

[0038] 1.1. Plasmid pUC57 (HBc-tG / M2 82-90 ) structure diagram

[0039] Plasmid pUC57-HBc-tG / M2 82-90 It is a plasmid vector comprising the chimeric coding gene sequence of the conserved antigen region of RSV G protein and the CTL epitope of M2 through codon optimization design and screening of the present invention. The protein encoded by the gene sequence is the antigen fragment (G144-204aa) (Plotnicky-Gilquin et al., 1999) of the G protein conserved B cell epitope encoded by RSV inserted between Asp78 and Pro80 of the HBc peptide, and the CTL epitope 82-90aa of RSV-encoded M2 protein (M2 82-90 ) fused to the C-terminus of the truncated protein HBc1-144aa, M2 82-90 The epitope can effectively activate the body's CD8 + T lymphocyte immune response (Srikiatkhachorn and Braciale, 1997). It is synthesized by a commercial company entrusted by the inventor, and the inte...

Embodiment 2

[0163] [Example 2] Large-scale preparation and verification of respiratory syncytial virus virus-like particles (VLPs) vaccine

[0164] 2.1. Large-scale preparation of respiratory syncytial virus-like particle (VLPs) vaccine

[0165] 1) Recombinant antigenic protein HBc-tG and HBc-tG / M2 82-90 a large number of expressions

[0166] (1) Take the frozen engineered bacteria out of the -80°C refrigerator, thaw, transfer 0.1ml of the bacteria liquid to 5ml of LB medium (containing kanamycin and chloramphenicol), and rotate at 220rpm at 37°C overnight.

[0167] (2) Inoculate the overnight culture into 1 LLB medium containing kanamycin and chloramphenicol at a ratio of 1:100, culture at 37°C with shaking at 220rpm. To be cultured to the logarithmic growth phase (bacterial solution OD 600 Value reaches 0.6), add IPTG to the final concentration of 0.2mM, 37°C, 220rpm rotating culture for 5 hours, induce protein expression.

[0168] (3) Transfer the induced culture to a 250ml centrif...

Embodiment 3

[0214] [Example 3] Functional detection of respiratory syncytial virus virus-like particles (VLPs) vaccine

[0215] Experimental protocol

[0216] Take 40 female BALB / c mice aged 5-6 weeks, and randomly divide them into 4 groups of A, B, C, and D, with 10 mice in each group. Before vaccination, blood is collected from the tail of the mice, and the serum is separated for antibody detection as negative control serum. Immunization was designed according to the following: Group A was injected with UV-inactivated RSV (1×10 5 TCID50); group B was injected with HBc-tG (VLPs), dose 20 μg / only; group C was injected with HBc-tG / M2 82-90 (VLPs), the dose is 20 μg / only. Vaccines were immunized by intramuscular injection, and each vaccine was immunized three times with an interval of three weeks; group D was the control group, which was injected with PBS by the same route and at intervals. Two weeks after immunization, blood was collected by tail docking and serum was separated. After...

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a respiratory syncytial virus (RSV) virus-like particle (VLPs) subunit vaccine as well as a preparation method and application thereof. The component of the vaccine is chimeric antigen protein which is fusion-expressed together with neutral antigenic epitope of an RSVG protein or simultaneously with the antigenic epitope of T cells of an M2 protein by taking hepatitis B virus core protein as a carrier. The high-purity antigen component is prepared by efficiently expressing antigen protein in escherichia coli and then performing in-vitro purification, degeneration and renaturation and self assembling into virus-like particles (VLPs). The RSVG protein contained in the VLPs and the antigenic epitope of the T cells of the M2 protein are simultaneously expressed, so that the capability of the vaccine for introducing specific immunity response and anti-RSV infection immunity protection can be enhanced, the balanced Th1 / Th2 immunity response can be induced and the RSV vaccine is prevented from enhancing the incidence of diseases. Animals are immunized and inoculated with VLPs vaccines to induce organisms to generate high-level RSV neutral antigens, enhanced Th1 cell factor level and effective protection on RSV attack infection.

Description

technical field [0001] The invention relates to a virus-like particle vaccine chimerically expressing multi-epitope antigens of respiratory syncytial virus and its preparation method and application, which belongs to the field of biotechnology and is used for the immune prevention of respiratory syncytial virus infection of humans and susceptible animals. Background technique [0002] Respiratory syncytial virus (Respiratory Syncytial Virus, RSV) is one of the important pathogens that cause lower respiratory tract infection in infants and young children worldwide and cause them to suffer from bronchiolitis and pneumonia, which can seriously cause bronchial asthma and even death. Almost all children before the age of two Have had at least one RSV infection (Black, 2003). The younger the age, the more severe the disease, the higher the reinfection rate, which is closely related to the occurrence of asthma; the immunocompromised are more susceptible to infection, and the clinic...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/70C12N1/21G01N33/569A61K39/155A61P31/14
Inventor 潘兹书乔磊柴枫
Owner WUHAN UNIV
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