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A kind of bivalent dna vaccine connection peptide and its application

A technology of DNA vaccine and connecting peptides, which is applied in the field of genetic engineering and immunology, can solve the problems of affecting the normal function of genes, not being able to protect men, and the imbalance of gene functions, so as to enhance vaccine immune response, enhance memory immune response, The effect of strong virus neutralization ability

Active Publication Date: 2015-09-30
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many ways to design linkers, most of which are neutral hydrophobic amino acids, such as polypeptide chains rich in glycine and serine. Its length is very important for protein folding and stability. The main problems of this method are: 1. The function of one or two genes may be affected due to the change of conformation; ②If the two gene products have different cellular localization, the normal function of the gene will also be affected, that is, the function of the gene is not balanced
[0005] There is no ideal vaccine for HSV-2 at present. The most commonly used method of producing vaccines is to use the envelope glycoprotein of the virus as an immunogen to make vaccines. The commonly used ones are gD and gB, but the neutralizing antibody produced by them is slow , The ability to neutralize the virus is low, the half-life is short, and it can only protect seronegative people and cannot protect men.

Method used

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  • A kind of bivalent dna vaccine connection peptide and its application
  • A kind of bivalent dna vaccine connection peptide and its application
  • A kind of bivalent dna vaccine connection peptide and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0041] The application of the bivalent DNA vaccine linking peptide in the preparation of the bivalent DNA vaccine is as follows:

[0042] 1. Gene amplification:

[0043] A. Construct HSV-2gD or gB successively on the pcDNA3.1 (+) (Invitrogen Company) plasmid vector (HSV-2 experimental strain is HG52 strain, obtains from British Royal Laboratory LGC Drug Impurity Standard Company) (GenBank: Z86099 .2) Envelope glycoprotein gene and adjuvant gene CCL19 (Kai Hu. JOURNAL OF IMMUNOLOGY, Aug. 2013, p. 1935-1947), its molecular formula is: pcDNA3.1(+)-HindⅢ-gD(EcoR I) / gB(BamH I) (to stop password) - (G4S) 2 -IZ-(G4S) 2 -CCL19 (remove signal peptide)-XhoI, named as p-gDIZCCL19 (for the pcDNA3.1 (+) recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO.3) and p-gBIZCCL19 (for the pcDNA3.1 (+) recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO.3) respectively pcDNA3.1 (+) recombinant plasmid with the nucleotide sequence shown in .6), and th...

Embodiment 2

[0064] The application of bivalent DNA vaccine in preparation intramuscular injection type vaccine, its step is:

[0065] 1. Expansion of plasmid culture: the bivalent DNA vaccine p-gDIZCCL19, p-CCL28IZgD, p-gBIZCCL19, p-CCL19IZgB and pcDNA3.1 (+) empty vector prepared above, and on pcDNA3.1 (+) alone Construction of antigen gD or gB (named p-gD or p-gB), separate construction of gene adjuvant CCL19 or CCL28 (named p-CCL19 or p-CCL28) plasmids (for monovalent vaccines) were retransformed into E.coli DH5α, Stretch and spread on agarose plate containing ampicillin resistance, culture at 37°C for 16 hours, pick a single colony and add it to 5mL liquid LB medium, shake at 200rmp at 37°C for 16 hours, then add 500mL at 1:100 Pre-prepared sterilized 2×YT liquid medium with ampicillin, cultured with shaking at 200rmp at 37°C for 20 hours, extracted the plasmid with a large amount of endotoxin-free plasmid extraction kit from MACHEREY-NAGEL company, and finally dissolved it in a mediu...

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Abstract

The invention discloses a bivalent DNA vaccine connecting peptide and applications thereof. A bivalent DNA vaccine is obtained through the following steps: performing PCR amplification on an HSV-2 glycoprotein gene and an adjuvant gene, introducing enzyme cutting sites, and introducing a connecting peptide (G4S)2IZ(G4S)2 via overlapping PCR. Humoral immunity, humoral immunity and mucosal immunity response reactions triggered by the bivalent DNA vaccine are detected by prime-boost principle immunized mice; a serotype antibody is detected and the severity of illness of each group within 15 days is evaluated by vaginal infection of150LD50 HSV-2. Compared with a univalent DNA vaccine, the recombinant bivalent DNA vaccine generates more intense humoral immunity, humoral immunity and mucosal immunity response reactions and a powerful capability of neutralizing the virus is detected out; high-titer antibodies can be rapidly generated by the immunity reactions and the anamnestic immunity reactions of the recombinant bivalent vaccine, so that a good protection effect on the mice is achieved.

Description

technical field [0001] The invention relates to the fields of genetic engineering and immunology, more specifically to a bivalent DNA vaccine linking peptide, and also relates to the application of the linking peptide in preparing a bivalent DNA vaccine. Background technique [0002] A bivalent DNA vaccine refers to a fusion DNA molecule that uses gene splicing technology and DNA recombination technology to connect two genes to form a fusion. After the target gene is introduced into the cell, it can be translated into a bifunctional protein molecule alone or in fusion, which can make the body produce specific vaccines. Biological agents for sexual immunity. [0003] The two genes are connected with a linker (linker) or directly, and the same open reading frame is used to translate a chimeric bifunctional fusion protein molecule. The general method is to delete the stop codon of the first gene, and its reading frame is the same as that of the first gene. The initiation codon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/79A61K48/00A61P31/12
CPCA61K39/00A61K48/00C12N15/62
Inventor 胡勤学阎岩胡凯
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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