Mucosa M-cell targeted viral myocarditis gene vaccine and preparation method thereof

A viral myocarditis and cell-targeting technology, applied in the field of biogenetic engineering, can solve the problems of low antigen expression, limited mucosal uptake and transport capacity, and inability to induce immune responses

Active Publication Date: 2013-10-09
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a kind of mucosal M cell-targeted viral myocarditis gene vaccine and its preparation, and said this mucosal M cell-targeted viral myocarditis gene vaccine and its preparation will solve the pr

Method used

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  • Mucosa M-cell targeted viral myocarditis gene vaccine and preparation method thereof
  • Mucosa M-cell targeted viral myocarditis gene vaccine and preparation method thereof
  • Mucosa M-cell targeted viral myocarditis gene vaccine and preparation method thereof

Examples

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Embodiment 1

[0064] Embodiment 1: Construction of pcDNA3.1-VP1B3 type Coxsackievirus gene vaccine

[0065]The RNA of the Nancy strain of Coxsackievirus type B3 in liquid phase was extracted according to the RNAex Reagent&System of Huashun Biological Co., Ltd., and the cDNA was synthesized according to the 2-N First Strand cDNA Synthesis Kit of Shanghai Sangon Biological Co., Ltd. Then, the DNA was used as a template and the VP1 upstream primer ( SEQ ID NO: 4) and the downstream primer (SEQ ID NO: 5) were amplified by CR to obtain the VP1 gene fragment, and at the same time, Hind III and BamHI restriction sites were respectively placed at both ends of the gene. Recover and purify the PCR product (Hua Shun Micro Gel Recovery Kit), and after double enzyme digestion, it can be connected into the corresponding vector pcDNA3.1(+). The ligation product was transformed into Escherichia coli DH5α, and the transformed colonies were screened with ampicillin. The pcDNA3-VP1 (pVP1) eukaryotic expressi...

Embodiment 2p

[0066] Example 2 Construction of pGEX-VP1 prokaryotic expression vector and protein expression and purification

[0067] The amplified pVP1 gene was double-digested with Xho I and Hind III, and connected with the prokaryotic expression vector pGEX cut by corresponding enzymes to construct the pGEX-VP1 prokaryotic expression plasmid.

[0068] Transform Escherichia coli BL21 (DE3) competent cells with pGEX-VP1, culture overnight at 37°C, and screen positive clones. Shake culture in LB (Amp100μg / ml) liquid medium until A600 reaches about 0.75, and add isopropylthiosemiglucoside (IPTG) to a final concentration of 0.5mM. Continue shaking and culturing for 3 hours, collect the cells by centrifugation at 4000r / min for 20min, resuspend in 1×PBS, and disrupt by ultrasonication. The supernatant was passed through an affinity chromatography column and eluted with eluents of different concentrations. Each 1ml of eluent was collected and the eluate with A280 greater than 1.0 was saved. Fi...

Embodiment 3

[0069] Example 3 Preparation of mucosal M cell targeted delivery system CPE30-chitosan

[0070] 1) Prepare a chitosan solution with a concentration of 0.1% by mass and a pH of 5.5. The molecular weight of chitosan is 390kDa, and the degree of deacetylation is 75% to 85%. The chitosan was dissolved in 1% HAc, stirred at 37°C to form a uniform colloidal solution of 1% chitosan, and then adjusted to pH 5.5 with 5mM sodium acetate solution to prepare a mass percent concentration 0.1% chitosan solution at pH 5.5.

[0071] 2) Preparation of deacetylated chitosan coupled with CPE30, using EDC / NHS as a coupling reagent to couple CPE30 to chitosan: First, chemically synthesize CPE30 polypeptide with N-terminal acetylation, N-terminal acetylation can be avoided Peptide-to-peptide coupling. Next, 200 μl of DMSO-dissolved CPE30 (1 mg), EDC (0.25 mg), and NHS (0.15 mg) were added to MES buffer (pH 6.0) and reacted at room temperature for 2 h. Then add β-mercaptoethanol (final concentra...

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Abstract

The invention discloses a mucosa M-cell targeted viral myocarditis gene vaccine. The vaccine is prepared by compounding a mucosa delivery system capable of targeting mucosa M-cells and B3-type coxsackie virus antigen encoding plasmids through cross-linking copolymerization. The invention further discloses a preparation method of the gene vaccine. The invention further discloses a preparation method of the mucosa delivery system capable of targeting the mucosa M-cells, and the mucosa delivery system is acquired after stable amide-ester bonds are formed by carboxyl groups and amino groups in deacetylated chitosan, wherein the carboxyl groups in peptide fragment CPE30 are targeted by M-cells which are activated by using EDC (Dichloroethane) and NHS (N-Hydroxysuccinimide). By nasally dropping the gene vaccine onto immunized mice, the gene vaccine is proved to be capable of effectively inducing the response between specific antigen serum and mucoantibody, obviously enhancing the local specific T-cell killing ability of the whole body and gastrointestinal mucosa and significantly improving the ability of mice for resisting B3-type coxsackie virus, thereby being an excellent prophylactic vaccine for viral myocarditis.

Description

technical field [0001] The invention relates to the field of biogenetic engineering, in particular to a vaccine for preventing or treating viral myocarditis and its preparation method and application, in particular to a mucosal M cell-targeted viral myocarditis gene vaccine and its preparation and application. Background technique [0002] Viral myocarditis (VMC) is a common clinical cardiovascular system disease, and its incidence is on the rise in recent years. In my country, the incidence rate of VMC is about 5%-15%. In the outbreak period, the incidence rate is as high as 30%-50%, and the case fatality rate reaches 24%. Not only that, recurrent attacks of VMC can lead to dilated cardiomyopathy and heart failure, and the 5-year mortality rate is as high as 75%, which has become a serious social health problem. Enteroviruses, especially coxsackievirus B3 (CVB3) type B3, are the main pathogens causing viral myocarditis. At present, there is a lack of clinically effective ...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/125A61K47/42C12N15/85C12N15/66C07K14/33C07K1/107A61P31/14
Inventor 熊思东岳艳徐薇叶婷
Owner SUZHOU UNIV
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