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Anti-melanoma cytotoxic lymphocyte (CTL) multi-epitope peptide and application thereof

An anti-melanoma and melanoma technology, applied in the field of medical biology, can solve the problems of difficult preparation, high cost of anti-melanoma peptide vaccines, and strong immune stimulation.

Inactive Publication Date: 2012-11-21
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

[0008] Aiming at the defects of current anti-melanoma peptide vaccines such as high cost, difficult preparation, and high immune stimulation intensity, the present invention constructs a combination of three restricted epitopes from two different antigens of MAGE-1 and MAGE-3 in series. Simple preparation, stable expression and high specificity multi-epitope polypeptide, and tumor nucleic acid vaccine containing the epitope polypeptide, so as to achieve the purpose of preventing and treating melanoma

Method used

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  • Anti-melanoma cytotoxic lymphocyte (CTL) multi-epitope peptide and application thereof
  • Anti-melanoma cytotoxic lymphocyte (CTL) multi-epitope peptide and application thereof
  • Anti-melanoma cytotoxic lymphocyte (CTL) multi-epitope peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Design and construction of CTL multi-epitope peptides

[0037] The purchased CTL multi-epitope peptides included 3 CTL epitopes, a Th universal epitope and 5 linkers. The three epitopes are CTL epitopes restricted by MHC class I molecules highly expressed on HCC, and they are:

[0038] MAGE-3 (271-279) FLWGPRALV (SEQ ID NO: 1),

[0039] AFP (542-550) GVALQTMKL (SEQ ID NO: 2)

[0040] MAGE-1 (278-286) KVLEYVIKV (SEQ ID NO: 3).

[0041] Then, according to computer-aided design optimization, each epitope and linker sequence are arranged in a beaded aggregation sequence. The principle guarantee is that each epitope is spaced with a suitable linker sequence to maintain the spatial conformation of each epitope without interfering with each other.

[0042] The amino acid sequence of the finally determined CTL multi-epitope peptide, the order from 5' to 3' is:

[0043] GAA-FLWGPRALV-NAAA-KVLEYVIKV-KAA-PADRE-GAAA

[0044] GVALQTMKL-GAA

[0045] Among them, FL...

Embodiment 2

[0051] Example 2: HLA-A*0201 affinity analysis of candidate peptides

[0052] MAGE-3 was synthesized separately (271-279) FLWGPRALV, AFP (542-550) GVALQTMKL and MAGE-1 (278-286) KVLEYVIKV 3 epitope polypeptides, the combination of polypeptides and HLA-A*0201 molecules was detected by immunofluorescence method. Using the fluorescence coefficient (FI) as the detection index of affinity, it was found that the three epitope peptides of the polypeptide HCC have high affinity with HLA-A*0201 molecules, see image 3 .

Embodiment 3

[0053] Example 3: Obtaining of HBc(1-75aa)-multi-epitope-HBc(75-144aa) chimeric gene sequence

[0054] At first with the DNA sequence of the epitope polypeptide of the present invention and the HBV (adr subtype) genomic DNA of whole gene synthesis respectively as template, amplify multi-epitope and HBc (1-75aa) (SEQ ID NO: 6) and HBc (75-144aa) (SEQ ID NO: 7) gene fragments, and then these three fragments are connected by PCR method to form a HBC (1-75aa)-multi-epitope-HBc (75-144aa) chimeric gene. Specific steps are as follows:

[0055] (1) Use primer P5 and primer P6 to amplify all genes encoding the multi-epitope sequence. Wherein primer P5 is: 5′-CGG GGCGCC TTTATGAACAAATTTATTTATG-3', the primer is consistent with the 5' end of the multi-epitope coding sequence, and simultaneously introduces a Bbe I restriction endonuclease site. Primer P6: 5′-GCG GAGCTC CGCCGCGCCCACCAG-3', introduces a Sac I restriction endonuclease site. PCR reactions were performed using high-fid...

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Abstract

The invention relates to the technical field of medical biology and provides an anti-melanoma cytotoxic lymphocyte (CTL) multi-epitope deoxyribose nucleic acid (DNA) vaccine. The coded gene of the vaccine comprises three CTL epitopes, a Th general epitope and five connexons, and the expressed amino acid sequence is shown as SEQ ID NO:5. The multi-epitope coded gene is connected with HBc(1-75aa) and HBc(75-144aa) by a polymerase chain reaction (PCR) method to form a mosaic gene; the mosaic gene and HLA-A*0201 / Kb fusion gene are respectively cloned to multiple cloning sites A and B of an eukaryotic expression vector pIRES so as to construct a coexpression vector; and the induced expression plasmids pc-HH of the fusion gene form annular double chains with the lengths of 9.3kb. The anti-melanoma CTL multi-epitope vaccine provided by the invention has the characteristics that the specificity of tumor cells can be stably expressed in vivo, and is simple to prepare, the preparation cost is low and the like.

Description

technical field [0001] The invention relates to the technical field of medical biology, in particular to the preparation of anti-melanoma CTL multi-epitope peptide and vaccine thereof by genetic engineering method. Background technique [0002] Melanoma is a malignant tumor arising from melanocytes in the skin, mucous membranes, eyes, and pigmented areas of the central nervous system. The tumor metastasizes early and spreads rapidly, and often the cancer cells have spread throughout the body before doctors make a diagnosis. Once it is assessed as a malignant tumor, it can die a few months after diagnosis, and it is currently the skin cancer with the highest known mortality rate. Surgical resection is traditionally used as the main treatment method, and the surgical effect is directly related to the histological type of the tumor, the depth of lesion invasion, and the presence or absence of lymph node metastasis. Since most of the patients were discovered late, it is genera...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N15/70C12N1/21A61K38/17A61K31/7088A61P35/00
Inventor 孙树汉单石郭瀛军章意亮王越刘音
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY