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Toxicity T cell position vaccine of the cell for treating Hepatitis B and the preparing method

A technology for cytotoxicity and hepatitis B, applied in antiviral agents, pharmaceutical formulations, antibody medical components, etc., can solve the problems of incomplete elimination of viruses and poor long-term curative effect, and achieve the effect of enhancing cellular immune response and promoting activation

Inactive Publication Date: 2007-09-19
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The treatment drugs for HBV infection currently mainly rely on IFN-α and nucleoside analogs, none of these drugs can completely clear the virus, and the long-term efficacy is poor

Method used

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  • Toxicity T cell position vaccine of the cell for treating Hepatitis B and the preparing method
  • Toxicity T cell position vaccine of the cell for treating Hepatitis B and the preparing method
  • Toxicity T cell position vaccine of the cell for treating Hepatitis B and the preparing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Expression of T cell epitope fusion protein

[0037] 1. Select CTL epitope

[0038] From HBV antigen CTL epitope data, 21 CTL epitopes and two general Th cell epitopes covering human leukocyte antigen (HLA) HLAA2, A3, B7 supertype and A2402 phenotype were selected.

[0039] The following are the CTL epitopes in the different HBV antigens selected in the present invention (the numbers indicate the amino acid position of the epitope corresponding to the HBV antigen, and the amino acid sequence and HLA restriction of the epitope are in parentheses.):

[0040] HBV pre-S2 antigen: preS2 109-123 (MQWNSTALHQALQDP, A3), preS2 152-161 (SILSKTGDPV, A3)

[0041] Surface antigen: S 177-185 (VLQAGFFLL, A2), S 183-191 (FLLTRILTI, A2), S 249-257 (LLCLIFLLV, A2), S 313-321 (IPIPSSWAF, B7), S 335-343 (WLSLLVPFV, A2), S 348-357 (GLSPTVWLSV, A2)

[0042] Core antigen: C 18-27 (FLPSDFFPSV, A2 / B7 / B51), C 117-125 (EYLVSFGVW, A2402), C 141-151 (STLPETTVVRR, A3)

[0043] Polymerase anti...

Embodiment 2

[0091] Example 2: Construction of immune adjuvant plasmid

[0092] 1. Amplification of CpG ODN vector DNA fragment

[0093] Using the eukaryotic expression plasmid vector pVAX1 (Invitrogen product) as a template, PCR amplifies the DNA fragments encoding the kanamycin resistance gene (KanR) and the pUC plasmid replication origin (pUC ori),

[0094] Upstream primer: 5'-GAATTCAAGCTTAGAGACAGGATGAGGATC-3',

[0095] Downstream primer: 5'-GAATTCGGATCC GTCAACGCGT ATATCTGG-3'.

[0096] The reaction conditions and procedures are the same as before.

[0097] 2. Connection of CpG ODN vector plasmid pKO

[0098] The amplified product obtained above was digested with endonuclease EcoRI, and the obtained DNA fragment was subjected to agarose gel electrophoresis, the target DNA band was recovered, and then self-circularized and ligated with T4 DNA ligase, and the ligated product was transformed into E. coli The resulting recombinant plasmid pKO. pKO contains KanR and pUC ori, and three single re...

Embodiment 3

[0108] Example 3: Preparation of vaccine

[0109] Purify the T cell epitope fusion antigen from yeast cells by hydrophobic chromatography and molecular sieve methods. Purify the plasmid adjuvant from E. coli with a plasmid mass preparation system (product of Qiagen, Germany), and dissolve it in a certain buffer, such as phosphate buffer. Solution (pH 7.0~7.4), with certain compatibility and certain concentration, such as 40~80μg of T cell epitope fusion antigen and 100~500μg of plasmid adjuvant. Mix the two to be a candidate vaccine for animal testing. Detect its efficacy, pharmacological activity and possible side effects, analyze its immunogenicity, and evaluate its application prospects as a therapeutic hepatitis B vaccine.

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Abstract

The invention relates to bio-pharmaceutical engineer technology domain. At present, the therapeutic drugs for HBV infection mainly depend on IFN-Alpha and nucleotide analog, which can not kill virus thoroughly with worse remote therapeutic effect. The invention provides a cytotoxic T cell epitope vaccine for hepatitis B, comprising a fused polypeptide formed by connecting 21 CTL epitopes selected from CTL epitopes data of HBV antigen and two universal epitopes of auxiliary T lymphocyte. Saccharomyces Serevisiae is selected to express the fused antigen and mixes with immunologic adjvant. The said vaccine can activate and enhance the cellular immune response of patient with chronic HBV infection. The vaccine can also promote the immune elimination of HBV, and can be used for treatment of chronic hepatitis B.

Description

Technical field: [0001] The invention relates to the technical field of biomedical engineering, and is a hepatitis B therapeutic cytotoxic T cell epitope vaccine and a preparation method thereof. Background technique: [0002] Hepatitis B virus (HBV) is a hepatotropic DNA virus that is prone to chronic infection after infecting the human body. It is the first cause of liver cirrhosis and primary liver cancer in Southeast Asian countries and regions, including my country. The treatment drugs for HBV infection currently mainly rely on IFN-α and nucleoside analogues. None of these drugs can completely eliminate the virus, and the long-term efficacy is poor. Therapeutic vaccine is a specific immunotherapy that has emerged in the past decade. Animal and clinical studies have shown that vaccination of such vaccines can enhance the cellular immune response of the infected person (or animal), thereby inhibiting virus replication. Summary of the invention: [0003] The purpose of the pre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/29A61K39/39A61P1/16A61P31/20C12N15/62
Inventor 陈志辉赵平王路戚中田
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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