Double-target DNA vaccine and constructing method thereof

A DNA vaccine, dual-targeting technology, applied in recombinant DNA technology, DNA/RNA fragments, pharmaceutical formulations, etc., can solve the problem of lack of good reference to research strategies in different fields, low gene transfer efficiency, scholars only focusing on research fields, research strategies, etc.

Inactive Publication Date: 2012-11-07
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 2006; 12(44): 7118-25)., CpG motif (Coban C, et al.Effect of plasma backbone modification by different human CpG motifs on the immunogenicity of DNA vaccine vectors.J Leukoc Biol.2005; 78(3 ): 647-55.), etc., to improve the immune protection ability of DNA vaccines, but the synergistic effect is limited
2005; 101(1-3): 137-49) and nanoparticles (Balenga NA, et al. Protective efficiency of dendrosomes as novel nano-sized adjuvants for DNA vaccination against birch pollen allergy. J Biotechnol. 2006; 124(3) : 602-14) and other compounds as representatives of non-viral vectors have good biological safety, simple use and low cost, but the efficiency of gene transfer is low
The reason may be that relevant scholars only pay attention to their own research fields and research strategies, but do not make good use of research strategies in different fields.

Method used

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  • Double-target DNA vaccine and constructing method thereof
  • Double-target DNA vaccine and constructing method thereof
  • Double-target DNA vaccine and constructing method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, preparation and identification of Escherichia coli bacterial ghost (BG)

[0048] 1. Construction of expression plasmids for heat-induced expression of phage E protein

[0049] The E protein gene of PhiX174 bacteriophage is connected into pHH43 ( H K. et al. The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism. J Biol Chem 1999; 274: 12316-12322), the specific method is as follows:

[0050] After the cleavage gene cassette (E-box) (including temperature-sensitive transcriptional regulatory region (cI857), lambda promoter region, and E gene region) was connected in series, kpn I and EcoRV restriction sites were added upstream and downstream, respectively, and then expressed The type vector pHH43 was digested and ligated, and the identification and sequencing were correct. Specific method: Carry out double digestion of the E-box fragment and the prokaryotic expression vector pHH43 with kpn...

Embodiment 2

[0060] Embodiment 2, construct the eukaryotic expression vector of mouse invariant chain (mIi)

[0061] With plasmid pQE31-mIi (Bischof F, et al.Melms A.Specific treatment of autoimmunity with recombinant invariant chains in which CLIP is replaced by self-epitopes.Proc Natl Acad Sci U S A.2001;98(21):12168-73 ) as a template, using the PCR method to obtain the mIi coding gene (introducing Xho I and EcoR I restriction sites at the 5' ends of the upstream and downstream primers respectively when designing the PCR primer sequence, the specific sequence is

[0062] P1: 5'-GCG CTCGAG ATGACGGATCCGCATGCGAGCT-3' and

[0063] P2: 5'-CGC GAATTC GCAGGGTGACTTGACCCAG-3').

[0064] The method is: in a 50 μl reaction system, primers (1 μl each for forward and reverse primers), template (1 μl), high-fidelity DNA polymerase (0.25 μl), dNTP (2.5 mM, 4 μl), 10× amplification Adding buffer (5μl) and deionized water (36.75μl) were shaken and mixed, after centrifugation, pre-denaturation at 9...

Embodiment 3

[0066] Embodiment 3, construct the FMDV-VP1 vector of endogenous targeting

[0067] 1. Select and synthesize the dominant epitope of foot-and-mouth disease virus (FMDV)

[0068] FMDV is a single-stranded positive-strand RNA virus, and its structural protein VP1 has multiple B cell and T cell epitopes, which can induce a strong immune response; among them, VP1 proteins 21-40, 141-160, 200-213 The antigenic epitope has the strongest antigenicity, and all kinds of serotype FMDV have this epitope, so it is a very important DNA vaccine candidate sequence. It is planned to use the 21-40+141-160 and 141-160+200-213 sequences as targets to construct corresponding expression vectors.

[0069] 2. Build the carrier

[0070] Such as Figure 7 As shown, use pDSRed-mIi as the eukaryotic expression vector (constructed in Example 2), use the 21-40+141-160, 141-160+200-213 sequences to replace the CLIP region of mIi, and construct a eukaryotic expression vector for VP1 Expression vector. Th...

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Abstract

The invention discloses a double-target DNA vaccine and a constructing method thereof. The vaccine uses BG (Butylene Glycol) as a DNA conveying carrier, and the DNA carries a DNA vaccine of a molecular chaperone constant chain Ii gene of MHC-II. The vaccine can prevent the degradation of the DNA vaccine and increase the expression level of a DNA coding sequence of the vaccine in an antigen presenting cell and can be used for further presenting the expressed antigen to an immunize effect cell efficiently, thereby greatly improving the immunocompetence of the DNA vaccine. The invention plays animportant role in the research (of improving the immunocompetence of the traditional DNA vaccine and the future DNA vaccine) of the novel vaccine and the control of future diseases, and has wide application prospect.

Description

technical field [0001] The invention relates to a vaccine and a construction method thereof in the field of biotechnology, in particular to a dual-target DNA vaccine and a construction method thereof. Background technique [0002] Vaccines are an effective way to prevent infectious diseases. So far, more than 80% of the children in the world have received 6 kinds of vaccines including diphtheria, measles, whooping cough, polio, tetanus and tuberculosis, which has greatly reduced the mortality rate of these diseases. But for many diseases, there are still many difficulties in the development and use of vaccines. Taking tuberculosis as an example, BCG vaccination only has a protective effect on 50%-80% of children, and the protection for adults is even lower. Cholera once caused a serious pandemic. In recent years, the number of cases has increased, and the commonly used Vibrio cholerae inactivated vaccine is only valid for 4 months. In addition, there are many difficulties...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K39/135A61K39/29A61K39/12C12N15/11C12N15/85A61P31/14A61P31/20A61P31/18
Inventor 季守平靳小攀檀英霞王颖丽李素波高红伟鲍国强宫锋
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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