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CVB3 myocarditis recombinant protein vaccine targeting draining lymph node and preparation method thereof

A recombinant protein and lymph node technology, applied in the field of genetic engineering, can solve the problems of difficult enrichment and presentation, easy degradation, etc., and achieve the effect of preventing viral myocarditis and improving the effect of immune protection.

Active Publication Date: 2019-10-29
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the shortcomings of CVB3 VP1 protein vaccines such as easy degradation and difficult enrichment and presentation in draining lymph nodes. By introducing bifunctional adjuvant molecules aimed at prolonging the retention of antigens in the body and efficiently targeting draining lymph nodes, the efficacy of the protein vaccine can be effectively improved. immune protection effect

Method used

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  • CVB3 myocarditis recombinant protein vaccine targeting draining lymph node and preparation method thereof
  • CVB3 myocarditis recombinant protein vaccine targeting draining lymph node and preparation method thereof
  • CVB3 myocarditis recombinant protein vaccine targeting draining lymph node and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1: Preparation of ABD-VP1 recombinant protein vaccine

[0040] (1) Construction of prokaryotic expression plasmid encoding CVB3 VP1

[0041] Using Coxsackievirus type B3 Nancy strain DNA as a template, use the VP1 upstream primer shown in SEQ ID NO: 7 and the VP1 downstream primer shown in SEQ ID NO: 8 to amplify the full-length coding gene of CVB3 dominant antigen VP1 by PCR method (The amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4);

[0042] The target gene was digested by Hind III and BamH I and connected to the pET28a vector which was also digested by the same double enzymes to obtain the prokaryotic expression plasmid (pET28a-VP1) encoding CVB3 VP1;

[0043] (2) Construct the prokaryotic expression plasmid encoding the ABD-VP1 protein vaccine

[0044] The full-length gene encoding ABD was chemically synthesized and constructed into plasmid pUC57 to obtain pUC57-ABD plasmid. Using this plasmid as a tem...

Embodiment 2

[0054] Embodiment 2: Characteristic detection of ABD-VP1 recombinant protein vaccine

[0055] 1) Binding test of ABD-VP1 and mouse serum albumin (MSA)

[0056] After protein purification, we tested the binding ability of the two proteins to MSA by ELISA method. The specific steps are as follows: first, coat the plate with mouse serum albumin (MSA) and bovine serum albumin (BSA), add 10 μg / ml, 100 μl / hole into a 96-well Elisa plate, overnight at 4°C; wash with 0.05% PBST After three times of blocking with 5% milk, 100 μl / well, incubate at room temperature for 1 h; wash three times with 0.05% PBST, add ABD-VP1 / VP1 (1 μg / ml) to the corresponding well, 100 μl / well, incubate at 37°C for 2 h; 0.05% PBST Wash three times with PBST, add VP1 antibody (1:8,000 dilution), 100μl / well, incubate at room temperature for 1h; wash three times with 0.05% PBST, add HRP-goat anti-rabbit IgG antibody (1:6,000 dilution), 100μl / well, incubate at room temperature 1h; wash five times with 0.05% PBST...

Embodiment 3

[0061] Embodiment 3: ABD-VP1 recombinant protein vaccine subcutaneously immunizes BALB / c mice

[0062] BALB / c male mice aged 6-8 weeks were divided into 3 groups: PBS, VP1, ABD-VP1, 6 mice in each group. A total of three immunizations were performed, and blood was collected from the posterior canthus vein every other week. The serum was collected at 37°C for 30 minutes and centrifuged at 3,000 rpm for 30 minutes. The serum was collected and frozen at -80°C for subsequent testing.

[0063] Immunization methods are as follows:

[0064] 1) Draw 25 μg recombinant protein (dissolved in 200 μl PBS) with a syringe and directly inject it into the subcutaneous part of the mouse,

[0065] 2) Take blood after one week,

[0066] 3) Repeat the immunization process of 1)+2) two weeks later,

[0067] 4) A total of 3 times of immunization, the total amount of VP1 protein is about 50-100 μg.

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Abstract

The invention discloses a CVB3 myocarditis recombinant protein vaccine targeting a draining lymph node and a preparation method thereof. Difunctional adjuvant molecules which aim to prolong antigen in-vivo retention and efficiently target the draining lymph node are introduced to effectively improve the immune protection effect of the protein vaccine. A serum albumin combination structural domainof streptococcus G protein and VP1 are recombined to prepare the ABD-VP1 recombinant protein vaccine, and the long-acting myocarditis recombinant protein vaccine is constructed to effectively activatebody CVB3 specific immune response and prevent viral myocarditis. The viral myocarditis recombinant protein vaccine ABD-VP1 immunizes a mouse in a subcutaneous way, antigens can be effectively enriched in the draining lymph node, specific antibody and T cell immunologic response for CVB3 is effectively induced, the viral myocarditis is effectively prevented, and therefore the vaccine is an excellent alternative protein vaccine for preventive viral myocarditis.

Description

technical field [0001] The invention relates to a CVB3 myocarditis recombinant protein vaccine targeting drainage lymph nodes and a preparation method thereof, belonging to the technical field of genetic engineering. Background technique [0002] Viral Myocarditis (VMC) is a localized or diffuse inflammatory lesion of the myocardium, with an incidence rate of about 5%-15%. It is also the leading cause of sudden death in adolescents. In recent years, the incidence of viral myocarditis has been increasing year by year, and it has become an important social public health problem that affects people's health and causes a sharp decline in the social labor force. Enteroviruses, especially Coxsackievirus B3 (CVB3), are the main pathogens that cause viral myocarditis. At present, although some progress has been made in the pathogenesis of CVB3 and the diagnosis and treatment of viral myocarditis, there is still a lack of effective vaccines and specific drugs for the prevention and...

Claims

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Application Information

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IPC IPC(8): A61K39/125A61K39/39A61P31/14
CPCA61K39/12A61K39/39A61P31/14C12N2770/32334A61K2039/55516A61K2039/6068Y02A50/30
Inventor 岳艳熊思东高月
Owner SUZHOU UNIV
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