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Preparation and application of Her2-neu antigen positive tumor therapeutic vaccine

A her2-neu and tumor antigen technology, which is applied in the field of preparation of Her2-neu antigen-positive tumor therapeutic vaccines, can solve the problems of not achieving satisfactory immune effects, inability to effectively induce immune responses, and the like

Active Publication Date: 2012-12-26
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 design and synthesis of methanol yeast codon-optimized sequence SEQ ID NO: 1, and a Not I restriction site was added at the 3' end, the sequence was connected 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 plasmids

[0103] 1. Her2 342-456 and the acquisition of DNA fragments of Hsp70L1:

[0104]Using the plasmid puc18-HER2 as a template, and using M13R and HSP-HER2-F as primers, the encoding Her2 used to construct the fusion protein was obtained 342-456 DNA fragment (PCR product A, SEQ ID NO: 1).

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

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

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

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

[0109] At the same time, collect the HeLa cells (ATCC Number: CCL-2) that were heat-induced at 41°C for 1 h, use the total cellular RNA extraction reagent Trizol (Invitrogen Company) to prepare the total cellular RNA, and use AMV reverse transcriptase (Promega Company) to synthesize the first chain, using it as a template, amplified with the following α-factor and ...

Embodiment 3

[0120] High expression of Hsp70L1-Her2 342-456 Construction of Engineering Bacteria and Identification of Positive Yeast Transformants

[0121] Kit (Qiagen) to extract a large amount 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, 200Ω to obtain yeast transformants.

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

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

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Abstract

The invention provides preparation and application of a Her2-neu antigen positive tumor therapeutic vaccine, and particularly provides a fusion protein. The fusion protein contains (a) a tumor antigen Her2-neu extracellular region element and (b) a heat shock protein element. The invention also provides a dendritic cell sensitized by fusion protein and a corresponding tumor therapeutic vaccine. Test proves Her2-neu antigen specific immunological response can be effectively activated by applying the vaccine in vivo, and the vaccine is effectively applied to Her2-neu positive tumor.

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 and 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 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 residu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12N1/19C12N5/0784C12P21/02A61K39/00A61K38/17A61K47/48A61P35/00
Inventor 吴艳峰万涛曹雪涛
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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