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Preparation and application of her2-neu antigen-positive tumor therapeutic vaccine

A her2-neu, positive technology, applied in the fields of biology and medicine, can solve the problems of not achieving satisfactory immune effect and effectively inducing immune response

Active Publication Date: 2017-10-17
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

With the discovery of T cell epitopes in the amino acid sequence of HER2 / neu and confirming that they can induce specific cellular immune responses in vitro, tumor vaccines based on these epitope peptides have attracted the attention of researchers. Disis adopted a complete P185 protein cannot effectively induce immune response, Georg used HLA-A*0201 restricted nonapeptide epitope E75 (Her2 369-377 ) as a peptide vaccine can induce a specific immune response in some disease-free patients who have received treatment for node-positive breast cancer (NPBC), however, no satisfactory immune effect has been achieved so far

Method used

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  • Preparation and application of her2-neu antigen-positive tumor therapeutic vaccine
  • Preparation and application of her2-neu antigen-positive tumor therapeutic vaccine
  • Preparation and application of her2-neu antigen-positive tumor therapeutic vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Her2 sequence design and optimization

[0100] According to the Her2 nucleic acid sequence, the methanol yeast codon optimal sequence SEQ ID NO:1 was designed and synthesized, and the Not I restriction site was added at the 3'end. The sequence was ligated to the T vector pUC18 (purchased from Invitrogen), denoted as puc18-HER2 . The amino acid sequence of the Her2 / neu extracellular region element encoded by the optimized nucleotide sequence (SEQ ID NO: 1) is shown in SEQ ID NO: 2, with a length of 115, corresponding to positions 342-456.

Embodiment 2

[0102] Construction of recombinant expression plasmid

[0103] 1.Her2 342-456 And Hsp70L1 DNA fragments:

[0104] Use plasmid puc18-HER2 as template, and M13R and HSP-HER2-F as primers to obtain the encoding Her2 for constructing the fusion protein 342-456 DNA fragment (PCR product A, SEQ ID NO: 1).

[0105] The sequence of the 5'end oligonucleotide primer M13R used in the PCR reaction is:

[0106] 5'-CAGGAAACAGCTATGACC-3', (SEQ ID NO: 3).

[0107] The sequence of the 3'end oligonucleotide primer HSP-HER2-F is:

[0108] 5'-CTCTATTGAGATAGCATCTTGTTACGGTTTGGGTATG-3' (SEQ ID NO: 4).

[0109] At the same time, collect HeLa cells (ATCC Number: CCL-2) heat-induced at 41°C for 1 h, and use the cell total RNA extraction reagent Trizol (Invitrogen) to prepare total cell RNA, and use AMV reverse transcriptase (Promega) to synthesize the first Chain, using it as a template, using the following sequence of α-factor and HSP-HER2-R as PCR oligonucleotide primers to amplify to obtain a DNA fragment enco...

Embodiment 3

[0120] Highly expressed Hsp70L1-Her2 342-456 Construction of engineering bacteria and identification of positive yeast transformants

[0121] Kit (Qiagen Company) for mass extraction of pPIC9k-Hsp70L1-Her2 342-456 The plasmid was linearized with SacI endonuclease and electrotransformed into GS115 competent yeast cells under the transformation conditions of 2.0kV, 25μF, and 200Ω to obtain yeast transformants.

[0122] The single clones on the MD plate were respectively inoculated into 3ml YPD medium, cultured at 30°C for 24 hours, and then the yeast genomic DNA was extracted, using α-factor primer (SEQ ID NO: 4) and 3'-aox primer (3'-AOX). : 5'-GCAAATGGCATTCTGACATCC-3', (SEQ ID NO: 7)), the yeast transformants were identified by PCR. Positive recombinants were identified by electrophoresis.

[0123] The results showed that for the positive yeast transformants (denoted as pPIC9k-Hsp70L1-Her2 342-456 Yeast), a PCR band of about 2.1k was obtained ( figure 2 ), consistent with the predi...

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Abstract

The invention provides the preparation and application of a Her2-neu antigen-positive tumor therapeutic vaccine. Specifically, the present invention provides a fusion protein, which contains (a) tumor antigen Her2-neu extracellular domain element and (b) heat shock protein element. The invention also provides the dendritic cells sensitized with the fusion protein and the corresponding tumor therapeutic vaccine. Tests show that the vaccine of the present invention can effectively stimulate Her2-neu antigen-specific immune response in vivo, and can be effectively applied to Her2-neu positive tumors.

Description

Technical field [0001] The present invention relates to the fields of biology and medicine. More specifically, the present invention relates to the preparation and application of a Her2-neu antigen-positive tumor therapeutic vaccine. The fusion protein includes heat shock protein and Her2-neu tumor antigen. Background technique [0002] HER2 / neu is an oncogene that often changes in human tumors. It is closely related to the occurrence and development of tumors. It is the second member of the epidermal growth-factor receptor (EGFR) family. The epidermal growth factor receptor family, also known as the HER or ErbB2 family, plays an important role in cell signal transduction and is an important regulator of cell growth, differentiation and survival. [0003] The human HER2 / neu gene is located on the short arm of chromosome 17, and the expression product is a single-chain transmembrane glycoprotein with a molecular weight of 185KDa, namely p185. p185 contains 1255 amino acid residues....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12N1/19C12N5/0784C12P21/02A61K39/00A61K39/39A61P35/00
Inventor 吴艳峰万涛曹雪涛
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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