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Nucleic acid vaccine adjuvant designed based on MCP-1 and construction method thereof

A MCP-1, nucleic acid vaccine technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problem of no nucleic acid vaccine adjuvant research reports and other problems

Inactive Publication Date: 2011-05-04
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

[0005] However, among the existing nucleic acid vaccine adjuvants, there are no research reports and related patents on nucleic acid vaccine adjuvants designed based on MCP-1

Method used

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  • Nucleic acid vaccine adjuvant designed based on MCP-1 and construction method thereof
  • Nucleic acid vaccine adjuvant designed based on MCP-1 and construction method thereof
  • Nucleic acid vaccine adjuvant designed based on MCP-1 and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: Construction of pcDNA.3.1-MCP-1 plasmid

[0030]First, according to the Internet database information (http: / / www.ncbi.nlm.nih.gov / gene / 20296), the mouse MCP-1 molecular sequence (Gene ID: 20296) was searched, and PCR primers were designed for its flanking genome coding sequence (Xbal I and EcoR I restriction sites were added upstream and downstream, respectively), using genomic DNA extracted from mouse peripheral blood mononuclear cells for PCR, the MCP-1 molecular sequence and PCR primers for cloning are shown in the table below. Thus, a DNA fragment containing the coding sequence of MCP-1 molecule was obtained.

[0031]

[0032] The DNA fragment comprising the pcDNA.3.1-MCP-1 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 MCP-1 expre...

Embodiment 2

[0033] Example 2: In vitro expression ability detection experiment under the condition of co-transfection of pcDNA3.1-MCP-1 plasmid and pVAX1-HBsAg nucleic acid vaccine

[0034] Using mice as an experimental animal model, inject pcDNA3.1-MCP-1 plasmid into the legs of mice, and use immunohistochemistry to analyze the recruitment of DC in the injection site 24 hours later, and found that pcDNA3.1-MCP-1 plasmid Compared with the control group after immunization, the immunization group had significant DC recruitment and activation.

Embodiment 3

[0035] Example 3: In vivo immune efficacy detection experiment after co-immunization of pcDNA3.1-MCP-1 plasmid and pVAX1-HBsAg nucleic acid vaccine

[0036] Immunize 6-8 week-old C57BL / 6J mice with 0.1mg / mouse of pcDNA3.1-MCP-1 and 0.1mg / mouse of pVAX1-HBsAg, immunize 3 times in total with 2-week intervals, and recombine with HBsAg at the end of the 6th week Protein (10 μg / cause) was mixed with incomplete Freund's adjuvant to boost immunization once. In terms of humoral immunity, ELISA was used to detect the antibody titer and IgG subtype (see figure 2 ); In terms of cellular immunity, the ELISPOT method was used to detect the secretion of IFN-γ in spleen T cells, and the in vitro induced spleen CTL target killing assay was used to detect antigen-specific CD8 + number of T cells. The HBsAg nucleic acid vaccine pVAX1-HBsAg was used as a parallel control, normal saline was used as a blank control, and the HBsAg subunit protein vaccine was used as a positive control. The resul...

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Abstract

The invention relates to the technical field of medical biology and discloses a plasmid for enhancing cellular immune effect of a nucleic acid vaccine, which is designed based on monocyte chemoattractant protein-1 (MCP-1). An expressed sequence of a novel MCP-1 spliceosome molecule is inserted into a pcDNA-3.1 plasmid to form a pcDNA-3.1-MCP-1 plasmid, the plasmid can efficiently express the novel MCP-1 spliceosome molecule, and the expression ability of the co-transfected nucleic acid vaccine is not influenced. In an in vivo test, the plasmid and the nucleic acid vaccine are injected simultaneously to have the capacity of effectively inducing cellular immune response, namely antigenic specificity spleen interferon (IFN)-gamma positive cells are increased and target-killing capacity of spleen CD8 positive cells is improved. Therefore, the plasmid can be added into the conventional nucleic acid vaccine preparation for mixed injection so as to obviously accelerate the nucleic acid vaccine-induced cellular immune response, and the plasmid serves as a novel cellular immune adjuvant.

Description

technical field [0001] The invention relates to the technical field of medical biology, in particular 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 corresponding immune protection . 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, sterilization, etc. The third generation of vaccines af...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39C12N15/19C12N15/63
Inventor 孙树汉章意亮郭灜军魏伟周奇王越
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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