Malaria infrared stage DNA vaccine and preparing method thereof

A DNA vaccine and infrared technology, which is applied in the field of biomedicine, can solve the problems that the efficiency and duration of immune protection cannot reach the effective malaria vaccine, and achieve the effect of good application prospects.

Inactive Publication Date: 2011-08-31
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing malaria infrared stage subunit vaccines mainly based on CSP cannot induce anti-CSP antibody production and CD8 + Efficiency and duration of T cell responses, immune protection did not meet WHO criteria for an effective malaria vaccine

Method used

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  • Malaria infrared stage DNA vaccine and preparing method thereof
  • Malaria infrared stage DNA vaccine and preparing method thereof
  • Malaria infrared stage DNA vaccine and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, construction of recombinant plasmid pFLAG-CMV8-gp96NTD / CSP

[0035] The schematic diagram of the construction of the recombinant plasmid pFLAG-CMV8-gp96NTD / CSP is as follows figure 1shown. It can be seen from the figure that pFLAG-CMV8-gp96NTD / CSP is the coding gene (nucleotide sequence shown in SEQ ID No.2) of gp96NTD (amino acid sequence shown in SEQ ID No.1) and CSP (amino acid sequence shown in SEQ ID No. ID No.3) coding gene (nucleotide sequence shown in SEQ ID No.4) is inserted in series into the multiple cloning site of the eukaryotic expression vector pFLAG-CMV8 not I and Bam H between Ⅰ and obtained.

[0036] 1. Construction of recombinant plasmid pMD19 simple T / CSP

[0037] 1. Isolation of Plasmodium sporozoites and extraction of total RNA

[0038] The BY265 strain of Plasmodium yoelii was infected with Anopheles stephensi according to the conventional method. On the 15th day after infection, the sporozoites were isolated from the saliva...

Embodiment 2

[0071] Example 2. Construction of recombinant plasmid pFLAG-CMV8-gp96 / CSP (control plasmid)

[0072] The schematic diagram of the construction of the recombinant plasmid pFLAG-CMV8-gp96 / CSP is as follows figure 2 shown. It can be seen from the figure that pFLAG-CMV8-gp96 / CSP is the heat shock protein gp96 polypeptide fragment gp96 ΔKDEL (The amino acid sequence is shown in SEQ ID No.9, that is, the key sequence KDEL anchored in the endoplasmic reticulum is removed from the full-length gp96 sequence) The coding gene (nucleotide sequence is shown in SEQ ID No.10) and The coding gene (nucleotide sequence shown in SEQ ID No.4) of CSP (amino acid sequence shown in SEQ ID No.3) is inserted into the multiple cloning site of eukaryotic expression vector pFLAG-CMV8 after tandem not I and Bam H between Ⅰ and obtained.

[0073] 1. Construction of recombinant plasmid pFLAG-CMV8-gp96

[0074] According to the gene sequence of gp96 (GenBank accession number U01153), PCR primers wer...

Embodiment 3

[0079] Example 3. Construction of recombinant plasmid pFLAG-CMV8-gp96NTD / peptide (control plasmid)

[0080] The schematic diagram of the construction of the recombinant plasmid pFLAG-CMV8-gp96NTD / peptide is as follows image 3 shown. It can be seen from the figure that pFLAG-CMV8-gp96NTD / peptide combines the coding gene (nucleotide sequence shown in SEQ ID No.2) of gp96NTD (amino acid sequence shown in SEQ ID No.1) with CSP-specific CD8 + T-cell epitope peptide (amino acid sequence shown in SEQ ID No.13) encoding gene (nucleotide sequence shown in SEQ ID No.14) was inserted into the multiple cloning site of eukaryotic expression vector pFLAG-CMV8 after tandem Bgl II and Bam H between Ⅰ and obtained.

[0081] 1. Encoding CSP-specific CD8 + Annealing of T cell epitope peptide oligonucleotides

[0082] Dissolve the CSP-specific CD8 with 1×T4 DNA ligase buffer + The sense and antisense chain oligonucleotides of T cell epitope peptide were lyophilized to a final concen...

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Abstract

The invention belongs to the biomedicine field, relating to a malaria infrared stage DNA vaccine and a preparing method thereof. The vaccine is obtained by connecting the coding gene of N end segment (amino acid sequence shown as SEQ ID No. 1) of heat shock protein (gp96) with the coding gene of the circumsporozoite protein (amino acid sequence shown as SEQ ID No. 3) in series and inserting into eukaryotic expression plasmid. The preparing method of the invention comprises three steps: the construction of recombinant plasmid pMD19 simple T/CSP, the construction of recombinant plasmid pFLAG-CMV8-gp96NTD, and the construction of recombinant plasmid pFLAG-CMV8-gp96NTD/CSP. The vaccine is able to simultaneously induce anti-CSP antibody generation and CD8+T cell reaction, thereby obtaining the plasmodium-specified complete protective immunization, and has great application prospect in preventing malaria inflection.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a malaria vaccine and a preparation method thereof. Background technique [0002] Malaria remains one of the most serious infectious diseases worldwide. Due to the emergence and spread of Plasmodium drug resistance and mosquito vector drug resistance, the drug control of malaria is facing difficulties, and the development of new, efficient and safe malaria vaccines has become an urgent need. [0003] There are three main types of malaria vaccines: red-phase vaccine, mosquito-phase transmission-blocking vaccine and infrared-phase vaccine. The currently effective malaria vaccine is only the attenuated sporozoite infrared phase vaccine, which can induce long-term effective protective immunity in mice, orangutans and humans, and the obtained protective immunity is species-specific rather than strain-specific, so it is not affected by malaria. Limitations of genetic background. Although the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/015C12N15/62C12N15/63
CPCY02A50/30
Inventor 徐文岳周桃莉
Owner ARMY MEDICAL UNIV
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