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Nucleic acid vaccine adjuvant and construction method thereof

A nucleic acid vaccine and construction method technology, applied in the field of medical biology, immune activity enhancement of nucleic acid vaccines, and can solve problems such as weak immune response

Inactive Publication Date: 2012-08-29
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there are also obvious shortcomings in DNA vaccines, that is, the ability of DNA vaccines to stimulate the body to generate immune responses is often weaker than that caused by conventional vaccination, which poses new challenges to the research of DNA vaccines

Method used

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  • Nucleic acid vaccine adjuvant and construction method thereof
  • Nucleic acid vaccine adjuvant and construction method thereof
  • Nucleic acid vaccine adjuvant and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of pcDNA3.1-Flt3L plasmid

[0029] We first searched the mouse Flt3L molecular sequence (GenBank: EU274583.1, www.pubmed.com) according to the Internet database information, and designed PCR primers for its flanking genomic coding sequence (adding Xbal I and EcoR I enzyme cutting sites to the upstream and downstream respectively) point), using the genomic DNA extracted from human peripheral blood mononuclear cells to carry out PCR, the Flt3L molecular sequence and PCR primers for cloning are shown in the table below. Thus, a DNA fragment containing the coding sequence of Flt3L molecule was obtained.

[0030]

[0031] The DNA fragment comprising the pcDNA.3.1-Flt3L coding sequence ( figure 1), using Xbal I and EcoR I restriction sites. The polyclonal restriction sites flanking the insertion sequence are still retained and can be used to insert the antigen sequence without affecting its effective expression. The above is the Flt3L expression ...

Embodiment 2

[0032] Example 2: Detection of expression ability in vitro under the condition of co-transfection of pcDNA3.1-Flt3L plasmid and pVAX1-HBsAg nucleic acid vaccine.

[0033] Using mice as an experimental animal model, the mice were injected with pcDNA3.1-Flt3L plasmid into their legs. After 24 hours, immunohistochemistry was used to analyze the DC recruitment in the injected area. Compared with the two groups, the immune group had significant DC recruitment and activation.

Embodiment 3

[0034] Example 3: Detection of immune efficacy in vivo after co-immunization of pcDNA3.1-Flt3L plasmid and pVAX1-HBsAg nucleic acid vaccine.

[0035] Immunize 6-8 week-old C57BL / 6 mice with 0.1mg / mouse of pcDNA3.1-Flt3L and 0.1mg / mouse of pVAX 1-HBsAg, immunize 3 times in total, with 2-week intervals, and use HBsAg recombinant protein at the end of the 6th week (10 μg / only) mixed incomplete Freund's adjuvant to boost immunization once, and in terms of humoral immunity, ELISA was used to detect the antibody titer (such as figure 2 shown); in terms of cellular immunity, the ELISPOT method was used to detect the IFN-γ secretion of spleen T cells, and it was found that (such as image 3 As shown), pcDNA3.1-Flt3L at 0.1 mg / monkey and pVAX1-HBsAg at 0.1 mg / monkey can effectively induce antigen-specific CTL response after immunization with 0.1 mg / monkey of pcDNA3.1-Flt3L. Increased, suggesting that antigen-specific cellular immunity was significantly enhanced.

[0036] Table 1. Ex...

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Abstract

The invention relates to the technical field of pharmaceutical biology. Flt3L is one of colony stimulating factor family members, and can be used for stimulating the proliferation and differentiation of CD34+hemopoietic stem cells. The invention aims to provide a nucleic acid vaccine adjuvant with the enhanced activity of cellular immunity and a construction method thereof. The nucleic acid vaccine adjuvant is a plasmid which is designed based on the Flt3L and is used for enhancing the cellular immune effect of a nucleic acid vaccine. In-vivo experiments prove that when simultaneously injected with the nucleic acid vaccine, the plasmid has the capacity of inducing a cellular immune reaction effectively, such as increasing of antigen-specific spleen IFN-gamma positive cells and enhancementof the target killing capacity of spleen CD8 positive cells. Therefore, the vaccine adjuvant of the invention can be added into the conventional nucleic acid vaccine preparation for mixing injection,so that the cellular immune reaction induced by the nucleic acid vaccine is promoted obviously, and the nucleic acid vaccine is used as a novel cellular immune adjuvant.

Description

technical field [0001] The invention relates to the technical field of medical biology. In this field, the present invention particularly relates to the technical field of enhancing the immune activity of nucleic acid vaccines. Background technique [0002] Nucleic acid vaccine refers to a plasmid vector containing a certain antigenic protein gene sequence as a vaccine, which is directly introduced into animal cells, and the antigenic protein is synthesized through the host's transcription system to induce the host to generate an immune response to the antigenic protein, so that the host can obtain the corresponding immunity. Protect. Because nucleic acid vaccines can induce a comprehensive immune response in the body, and have cross-resistance effects on different subtypes of pathogens, and at the same time have a series of advantages such as long-lasting immune protection effects, safety, and convenient production, they are considered to be secondary attenuation, steriliz...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/39A61K48/00C12N15/54
Inventor 孙树汉周奇郭瀛军张毅章意亮王越
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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