TB vaccine heat reversal protein65 and multiepi-position HER-2 Antigen fusion protein recombinat protein vaccine

A technology of heat shock protein and recombinant protein, which is applied in the vaccine for the prevention and treatment of human breast cancer, and in the field of genes encoding the vaccine, can solve the problems of inability to produce tumor prevention and treatment effects, and inability to effectively activate tumor CTL.

Inactive Publication Date: 2003-04-16
BEIJING HYDVAX BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the exogenous protein tumor antigen is immunized to the human body, it is usually taken up and processed by the antigen-presenting cells, enters the MHC class II presentation pathway, and stimulates the humoral immune response (Heikema A, Agsteribbe E, WilschutJ, Huckriede A. Generation of heat shock protein-based vaccines by intracellular loading of gp96 with antigenic peptides. Immunol Lett 1997 Jun 1; 57(1-3): 69-74), but can not effectively activate the production of tumor-specific CTL, thus can not produce effective tumor Preventive and therapeutic effects

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1 obtains the coding gene of BCG 65KD heat shock protein (HSP65)

[0021] BCG was obtained from Changchun Institute of Biological Products. Sutong potato medium is used to cultivate BCG at a temperature of 37-39° C., and the grown BCG presents dry wrinkled light yellow pellicle. The pellicles were collected from which BCG genomic DNA was extracted.

[0022] The method for extracting BCG genomic DNA refers to Molecular Cloning (J. Sambrook, Isolation of high-molecular-weight DNA from mammalian cells, 9.16-9.22, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989).

[0023] Heat shock protein 65 (HSP65) structural gene was isolated from BCG by PCR method. The 5' end primer sequence adopted is 5' CCATG GCC AAG ACA ATT GCG3', and the 3' (SEQ ID NO: 21) end primer sequence is 5' ACC GAA TTC GCT AGC CAT ATG GAA ATC CATGCC ACC CAT 3' (SEQ ID NO :twenty two).

[0024] The PCR operating procedure is: add the following reagents in a 500 μl microcentrifuge ...

Embodiment 2

[0035] Example 2. Synthetic multi-epitope HER-2 antigen gene

[0036] Using four rounds of PCR to synthesize the multi-epitope HER-2 antigen gene 1) The sequence of the first round of PCR primer 1 is: 5'TGACGGCGACCCGGCATCTAATACCGCACCGCTTCAACCGGAACAACTGCAAGTA TTTGAGACTCTGGAA3′ (SEQ ID NO:9) The sequence of primer 1' is: 5'TGTTAGCTTTCGGGGAGGTGTTTTCACGCAGAACCTTGATAGCAACGATTTC TTCCAGAGTCTCAAA 3′ (SEQ ID NO: 10) The sequence of the product synthesized by the first round of PCR is: 5'TGACGGCGACCCGGCATCTAATACCGCACCGCTTCAACCGGAACAACTGCAAGTATTTGAGACTCTGGAAGAAATCGTTGCTATCAAGGTTCTGCGTGAAAACACCTCCCCGAAAGCTAACA3' (SEQ ID NO: 11) 2) The sequence of the second round of PCR primer 2 is: 5'CTTCGGCTCCATCTCGAGCATTC TGACGGCGACCCGGC3' (SEQ ID NO: 12) The sequence of primer 2' is 5’ ACCAACTACAGCGGAGATGATAGAAGTCAGCGGTTCTT TGTTAGCTTTCGGGG3' (SEQID NO:13)产物的序列是:5’CTTCGGCTCTCTCGCATTCCTGCCAGAATCTTTTGACGGCGACCCGGCATCTAATACCGCACCGCTTCAACCGGAACAACTGCAAGTATTTGAGACTCTGGAAGAAATCGTTGCTATCAAGGTTCTGCGTGAA...

Embodiment 3

[0038] Example 3. Construction of BCG HSP-65 and multi-epitope HER-2 antigen gene fusion gene

[0039] The coding gene of BCG 65KD heat shock protein (HSP65) and the multi-epitope HER-2 antigen gene were respectively digested with EcoRI at 37° C. for 2 hours. Separation by agarose gel electrophoresis

[0040] products of digestion. The electrophoresis conditions are: 1% agarose gel, 1×TAE buffer solution, 150-200mA, electrophoresis for 0.5-1 hour.

[0041] 20×TAE buffer: 0.8mol / L Tris base

[0042] 0.4mol / L NaOAc

[0043] 0.04mol / L Na2EDTA

[0044] Adjust the pH to 8.3 with glacial acetic acid.

[0045] Observe and excise the DNA electrophoresis band on the agarose gel under ultraviolet light. Cut out the agarose gel containing the DNA band, freeze at -70°C for 15 minutes, and then melt the gel in a 65°C water bath; add an equal volume of TE-saturated phenol, shake vigorously for 30 seconds, and then -70°C Freeze at ℃ for 15 minutes; after thawing at room temperature, c...

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Abstract

The present invention discloses a recombinant protein vaccine which is a fusion protein prepared from the BCG vaccine heat-shock protein 65 and multi-epitope HER-2 antigen and can be used for effectively preventing and treating breast cancer. The gene for coding the said recombinant protein vaccine is also disclosed.

Description

field of invention [0001] The invention relates to a genetically engineered recombinant protein vaccine, in particular to a vaccine for preventing and treating human breast cancer. The invention also relates to the gene encoding the vaccine. Background of the invention [0002] Traditional breast cancer treatment methods include surgery, radiation therapy, chemotherapy, hormone therapy, and antibody therapy, but each of these methods has its own limitations. [0003] HER-2 protein (hereinafter referred to as HER-2) is encoded by the c-erbB-2 gene located on q21 of human chromosome 17. It is an active transmembrane glycoprotein with tyrosine kinase and epidermal growth factor The receptors have high homology (Tadashi Yamamoto, et al. Similarity of protein encoded by the human c-erbB-2 gene to epidermal growth factor receptor. Nature vol319 16 January 1986, 230-234). Amplification and overexpression of the gene encoding HER-2 can lead to transformation of cells. HER2 protei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61P35/00C12N15/62
Inventor 王丽颖王宣军于永利
Owner BEIJING HYDVAX BIOTECH
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