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Double-target DNA vaccine and constructing method thereof

A DNA vaccine and dual-targeting technology, applied in recombinant DNA technology, DNA/RNA fragments, pharmaceutical formulations, etc., can solve the problem that scholars only focus on research fields, research strategies, low efficiency of gene introduction, and failure to learn from different fields Research strategy and other issues

Inactive Publication Date: 2011-03-30
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

2005; 101(1-3): 137-49) and nanoparticles (BalengaNA. et al. Protective efficiency of dendrosomes as novel nano-sized adjuvants for DNA vaccination against birch pollen allergy. J Biotechnol. 2006; 124(3): 602 Non-viral vectors represented by compounds such as -14) have good biological safety, are simple to use, and low in 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

[0046] Embodiment 1, preparation and identification of Escherichia coli bacterial ghost (BG)

[0047] 1. Construction of expression plasmids for heat-induced expression of phage E protein

[0048] The E protein gene of PhiX174 bacteriophage is connected into pHH43 ( H K, et al. Therecombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism. JBiol Chem 1999; 274: 12316-12322), the specific method is as follows:

[0049] After the cleavage gene box (E-box) (including temperature-sensitive transcriptional regulatory region (cI857), lambda promoter region, E gene region) was connected in series, kpn I and EcoR V restriction sites were added upstream and downstream, respectively, and then and The expression vector pHH43 was digested and ligated, and the identification and sequencing were correct. For the sequence of the E gene cleavage cassette, see figure 2 . Specific method: Carry out double digestion of the E-box fra...

Embodiment 2

[0059] Embodiment 2, construct the eukaryotic expression vector of mouse invariant chain (mIi)

[0060] 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 US A.2001; 98(21):12168-73) as Template, using the PCR method to obtain the mIi coding gene (when designing the PCR primer sequence, introduce Xho I and EcoR I restriction sites at the 5' ends of the upstream and downstream primers respectively, and the specific sequence is

[0061] P1: 5'-GCG CTCGAG ATGACGGATCCGCATGCGAGCT-3' and

[0062] P2: 5'-CGC GAATTC GCAGGGTGACTTGACCCAG-3').

[0063] 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 94°C f...

Embodiment 3

[0065] Embodiment 3, construct the FMDV-VP1 vector of endogenous targeting

[0066] 1. Select and synthesize the dominant epitope of foot-and-mouth disease virus (FMDV)

[0067] 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.

[0068] 2. Build the carrier

[0069] Such as Figure 8 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 an important 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 Applications(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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