Hepatitis B virus (HBV) specific human leukocyte antigen-A33 (HLA-A33) restrictive epitope peptides and application thereof

A hepatitis B virus, hepatitis B technology, applied in the field of immunology, can solve the problems of disappearance, decreased T cells and cellular immunity, etc.

Inactive Publication Date: 2012-11-28
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Various viruses have different mechanisms for inducing immune tolerance, but eventually they all lead to the decline or disappearance of B cells’ ability to produce antibodies, T cells, and cellular immunity, either or both, which makes the virus persist

Method used

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  • Hepatitis B virus (HBV) specific human leukocyte antigen-A33 (HLA-A33) restrictive epitope peptides and application thereof
  • Hepatitis B virus (HBV) specific human leukocyte antigen-A33 (HLA-A33) restrictive epitope peptides and application thereof
  • Hepatitis B virus (HBV) specific human leukocyte antigen-A33 (HLA-A33) restrictive epitope peptides and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Construction of HLA_A33-restricted epitope peptide screening platform

[0038] 1: Construction of HLA_A3303 stable cell line:

[0039] In this example, the VSVG pseudovirus system (purchased from Biovector Science Lab, Inc) was used to add NheI and XhoI sites to both ends of the human HLA_A3303 gene (hereinafter referred to as human HLA_A3303, GenBank: U09740.1). , the sequence was fully synthesized in Shanghai Xuguan Biological Company (the nucleic acid sequence is SEQ ID No: 1, and its expression can produce the protein represented by SEQ ID No: 2), and then constructed in the vector plentilox using NheI and XhoI two sites 3.7-RRP (which is the plasmid in the above-mentioned VSVG pseudovirus system (purchased from Biovector Science Lab, Inc)) forms the recombinant plasmid plentilox-HLA_A33, extracts 15 μg of the recombinant plasmid, and then mixes it with 5 μg VSVG plasmid and 5 μg RSV / REV plasmid, 5 μg of pMDLG plasmids (the above three plasmids are also ...

Embodiment 2

[0046] Example 2: Screening of HBV-specific HLA_A33-specific epitopes

[0047] Using the following resources that have been published on the Internet, the possible candidate polypeptides of HBV subtypes B and C were predicted:

[0048] Three-dimensional quantitative structure-activity relationship program is MHCPred

[0049] The scoring matrix program is SYFPEITHI

[0050] The TAP program is TAPpred

[0051] According to the results of the three, 16 peptides were selected as candidate polypeptides. The overall results are shown in Table 1 below.

[0052]

[0053] According to the method of the above-mentioned peptide binding experiment, except that the peptide concentration at the time of screening was fixed at a final concentration of 50 μM, other methods remained unchanged, and the epitope peptides that could bind to HLA_A3303 among the predicted candidate peptides were screened.

[0054] figure 2 The cell screening results of peptides 1-5 and 7-8 of the present inv...

Embodiment 3

[0055] Example 3: Identification of candidate peptides by trimer renaturation

[0056] 1: HLA_A3303 Escherichia coli inclusion body expression and purification:

[0057] After optimizing the codon sequence and mRNA sequence of HLA_A3303 (the optimized coding nucleic acid sequence is SEQ ID No: 3), the optimized expression cassette was cloned into the vector pET30A (purchased from Novagen) with enzymes NdeI-XhoI to form The clone named pET30-HLA_A33HC-LND, its expression can produce the protein represented by SEQ ID No: 4, which contains α1, α2, α3 domains of HLA_A molecules and a biotinylation site sequence LND.

[0058] According to the conventional expression method, when the OD value of the Escherichia coli (E.coli) bacterial liquid reaches 0.7, add IPTG to make the final concentration 0.5mMol / L, harvest the bacteria after induction at 37°C for 4 hours, and use lysozyme (sigma) 5mg / L (per liter of original medium) treated at room temperature for 1 hour, then freeze-thawed ...

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Abstract

The invention relates to hepatitis B virus (HBV) specific human leukocyte antigen-A33 (HLA-A33) restrictive epitope peptides and application thereof. An HLA-A33 restrictive cell level epitope peptide screening platform is constructed; and 5 important HBV specific HLA-A33 restrictive epitope peptides (namely SEQ ID No: 5, 7, 9, 10 and 19) are determined by combining computer simulation analysis, detecting on the cell level epitope peptide screening platform and using three detection technologies of trimerical protein level renaturation, tetrameric patient's blood sample detection and Elispot detection. The invention also discloses the application of the screened HBV specific HLA-A33 restrictive epitope peptides in preparation of a medicament for treating hepatitis B in examinees, and specifically, the hepatitis B of the examinees is of HLA-A33 type. The invention also provides a medicament for treating the HLA-A33 hepatitis B in the examinees, and the medicament contains one or more of the 5 HBV specific HLA-A33 restrictive epitope peptides (namely SEQ ID No: 5, 7, 9, 10 and 19) and a medicinal carrier.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to a hepatitis B virus (HBV) specific HLA_A33 restricted epitope peptide and an application thereof. Background technique [0002] 1: Significance of discovering HLA_A33-restricted HBV-specific epitopes [0003] Hepatitis B virus (HBV) infection is still one of the most important global health problems. By the end of 2009, there were about 350 million chronically infected people in the world, and the number of people who died directly or indirectly from HBV infection every year up to 1 million. The proportion of HBV-infected people in my country is larger, estimated to be close to 10% of the total population. HBV infection can present a variety of clinical manifestations, including acute, chronic, and severe hepatitis according to the disease onset speed, as well as liver cirrhosis and liver cancer according to the development process, and eventually death due to liver failur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06A61K39/29A61P31/20
Inventor 田波高福潘煦文
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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