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Recombinant plasmid vaccine for treating hepatitis B and composition thereof

A technology of recombinant plasmids and plasmids, which is applied in the field of biomedicine and can solve the problem that the immune effect does not reach the ideal level.

Inactive Publication Date: 2013-06-12
BEIJING KAWIN TECH SHARE HLDG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Although the preliminary clinical trial results of DNA vaccine are satisfactory, its immune effect has not yet reached the ideal level. Therefore, how to enhance the immune effect has become the key to DNA vaccine research, especially how to ensure that DNA vaccine can simultaneously obtain a stronger effect. The immune effect of HBsAg and HBcAg

Method used

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  • Recombinant plasmid vaccine for treating hepatitis B and composition thereof
  • Recombinant plasmid vaccine for treating hepatitis B and composition thereof
  • Recombinant plasmid vaccine for treating hepatitis B and composition thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1 Construction of recombinant plasmid pRec2.0-preS2S-C (abbreviated as pS2S-C)

[0104] (1) First construct the recombinant plasmid vector backbone pOE-EKS, whose nucleotide sequence is the nucleotide sequence indicated by Seq No.1:

[0105] Using pDRVISV1.0 as a template, Primer1 and Primer2 as primers, among which primer 2 is a primer phosphorylated at the 5' end, amplified to obtain a replicon region (Ori) with a size of 748bp, and introducing EcoRI, KpnI and SwaI restriction sites at the same time point;

[0106] Primer1: 5'-GGAATTCGGGGTACCATTTAAATTTGAACGTTCGCAAtATGTGAGCAAAAGGCCAGC-3'

[0107] Primer2: 5'-CGGCGCGCGCCGAAAACGACGATTGCGAACGTTCAACCCGTAGAAAAAGATCAAAGG-3'

[0108] Using pDRVISV1.0 as a template, Primer3 and Primer4 as primers, among which primer 3 is a primer phosphorylated at the 5' end, amplified to obtain a Kanna resistance marker gene (Kan) with a size of 1056 bp, and introducing an EcoRI restriction site ;

[0109] Primer3: 5'-tcgtcgttttc...

Embodiment 2

[0129] The Core expression cassette was excised from the plasmid pcDNA3.1-C by BglII+PvuII double digestion, and cloned into pRec2.0-preS2S which was digested by BglII+EcoRV to construct the recombinant plasmid pRec2.0-PreS2S-C. Example 2 Construction of recombinant plasmid pRec2.0-C-preS2S (pC-S2S)

[0130] Using the pRec2.0-PreS2S constructed in Example 1 as a template, using Primer11 and Primer12 as primers, amplify the PreS2S gene with a size of 873bp, and pass HindIII+XbaI double digestion and HindIII+XbaI double digestion vector pcDNA3 .1 connect, construct and obtain pcDNA3.1-PreS2S;

[0131] Primer11:5'-CCCAAGCTTGCCGCCACCATGCAGTGGAACTC-3'

[0132] Primer12:5'-GCTCTAGAATCAGATGTAAACCCAC-3'

[0133] The PreS2S expression cassette was excised from the plasmid pcDNA3.1-PreS2S by BglII+PvuII double enzyme digestion, and cloned into pRec2.0-C which was digested by BglII+EcoRV double enzymes, and the recombinant plasmid pRec2.0-C-PreS2S (pC -S2S).

Embodiment 3

[0134] Example 3 Construction of recombinant adjuvant plasmid pmIL12

[0135] (1) The synthetic gene mP35 is connected with the carrier pcDNA3.1 of HindIII+XbaI double digestion by HindIII+XbaI double digestion, and the recombinant plasmid pcDNA3.1-mP35 is constructed and obtained;

[0136] (2) The mP35 gene expression cassette was cut out from the plasmid pcDNA3.1-mP35 by BglII+PvuII double digestion, and cloned into the plasmid pRec2.0-PreS2S which was digested by BglII+EcoRV to obtain the recombinant plasmid pRec2.0-PreS2S -mP35;

[0137] (3) Ligate the synthetic gene mP40 with the HindIII+XbaI double-digested vector pcDNA3.1 through HindIII+XbaI double-digestion to construct and obtain the recombinant plasmid pcDNA3.1-mP40;

[0138] (4) The mP40 gene expression cassette was cut out by SalI+PvuII double enzymes, and cloned into the plasmid pRec2.0-PreS2S-mP35 cut by SalI+SwaI double enzymes to obtain the recombinant plasmid pRec2.0-PreS2S-mIL12;

[0139] (5) Digest the pl...

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Abstract

The invention relates to a recombinant plasmid DNA (deoxyribonucleic acid) vaccine. The recombinant plasmid DNA vaccine carries a hepatitis B surface antigen protein encoding gene and a hepatitis B core antigen protein encoding gene. The constructed recombinant plasmid DNA vaccine comprises two target protein expression boxes, wherein one of the two target protein expression boxes is an optimized target protein expression box. The two genes are separated from each other by one target protein expression box. The invention further relates to a dual-plasmid DNA vaccine. The dual-plasmid DNA vaccine comprises the recombinant plasmid DNA vaccine and a recombinant interleukin-12 adjuvant plasmid. The constructed recombinant plasmid DNA vaccine and the composition thereof can be used for preparing a medicament for preventing and treating hepatitis B.

Description

technical field [0001] The invention relates to a recombinant plasmid DNA vaccine for treating hepatitis B disease and its composition, belonging to the field of biomedicine. Background technique [0002] Hepatitis B is a serious threat to human health, and there is no effective treatment at present. The main reason for the chronicity of hepatitis B is that the body lacks persistent specific cellular immunity and humoral immunity after hepatitis B virus (HBV) infection. [0003] HBV coat protein is composed of large protein LS (S1S2S antigen, encoded by pre-S1-pre-S2-S gene), medium protein MS (S2S antigen, encoded by pre-S2-S gene), small protein S (S antigen, encoded by S gene coding) composed of three molecules, among which the pre-S2 antigen has a small molecular weight and strong antigenicity, and is involved in the adsorption of hepatitis B virus to host cells. Therefore, the vaccine containing the pre-S2 antigen should be more effective in protecting the vaccinated. ...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/29A61K39/39A61P37/04A61P31/20C12N15/85C12N15/66C12N1/21C12R1/19
Inventor 李鼎峰刘勇周德胜张春丽
Owner BEIJING KAWIN TECH SHARE HLDG
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