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EGFR and HER2 combined polypeptide epitope vaccine

An epitope vaccine and epitope technology, applied in the field of medicine and biology, can solve the problems of single vaccine and limited effect, and achieve the effect of reducing immunogenicity and increasing safety

Inactive Publication Date: 2012-02-22
JIANGSU PROVINCE INST OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these vaccines are single, only targeting a certain member of the HER family, and have their own limitations on the effects of other HER family members

Method used

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  • EGFR and HER2 combined polypeptide epitope vaccine
  • EGFR and HER2 combined polypeptide epitope vaccine
  • EGFR and HER2 combined polypeptide epitope vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Construction of fusion protein gene using overlapping PCR technique.

[0056] Nucleotide sequence (SEQ ID NO.9) encoding HBcAg-△n protein, before the first amino acid codon, the start codon ATG and the recognition site of restriction endonuclease are added, and the last amino acid The codon is followed by a stop codon TAA and a recognition site for a restriction enzyme.

[0057] The fragment FI (99bp) containing three B epitopes was amplified by using primers P1 and P2 as templates. The PCR reaction reagents and conditions are as follows:

[0058]

[0059] Supplement H 2 0 to 50 μl reaction conditions are as follows:

[0060]

[0061] Cool to 4°C,

[0062] After the reaction, 1.0% Agarose electrophoresis was carried out to identify and tap the gel to recover the target band.

[0063] Fragment FII (116bp) was amplified by PCR using FI as template and P5 and P6 as primers. The PCR reaction reagents and conditions are as follows:

[0064]

[0065...

Embodiment 2

[0090] Example 2: Construction of recombinant expression vector pET28a / HBcAg-△ n .

[0091]Using the expression vector pET28a, the synthesized HBcAg-△n gene (the initial end contains the NcoI restriction site, the termination end contains the HindIII restriction site), and pET28a were respectively subjected to NcoI and HindIII double enzyme digestion treatment, the enzyme digestion conditions are Incubate at 37°C for 4 hours. Then the digested products were electrophoresed on 1% agarose gel for 15 minutes at a voltage of 120 volts. The above two DNA fragments were recovered with a gel recovery kit. The process is as follows: a. Cut off the agarose gel containing the target DNA under ultraviolet light, absorb the liquid on the surface of the gel with a paper towel and chop it up. Calculate the weight of the gel (record the weight of the 1.5ml centrifuge tube in advance), and use this weight as a gel volume (eg 100mg=100μl volume). b. Add 3 gel volumes of BufferDE-A, mix wel...

Embodiment 3

[0092] Example 3: Construction of recombinant bacteria.

[0093] The expression vector obtained in Example 2 was transformed into host cells to form transformed cells capable of producing recombinant HBcAg-Δn fusion protein, which was then detected.

[0094] The preparation of host cells refers to the preparation of competent cells of Escherichia coli BL21 (DE3) by a conventional calcium chloride method by using molecular cloning technology. Add 20 μl of the ligation solution (composition of the ligation solution: 15 μl of DNA fragments, 2 μl of carrier fragments, 1 μl of T4 ligase, 2 μl of 10-fold DNA ligation buffer, and a total volume of 20 μl) into 200 μl of competent cells. In bacteria, place on ice for 1 hour; heat shock at 42°C for 90 sec, quickly place on ice for 5 minutes; add 800 μl 37°C preheated LB culture solution; shake at 37°C, 220 rpm for 1 hour, centrifuge and spread all over the medium containing 50 μg / ml Kan LB plates were cultured overnight at 37°C upside ...

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Abstract

The invention discloses an EGFR and HER2 combined polypeptide epitope vaccine; two B cell epitope mimic polypeptide I and II of HER2 of the HER family, and a B cell epitope mimic polypeptide of EGFR of the HER family are inserted in a serial form between the 78th amino acid and the 79th amino acid of a hepatitis B core antigen HBcAg so as to form fusion protein which is the combined polypeptide epitope vaccine. The EGFR and HER2 combined polypeptide epitope vaccine of the invention breaks through the limitations of existing single epitope vaccines, and the combined epitope vaccine is providedwith more extensive antineoplastic activity.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and specifically relates to a kind of EGFR and HER2 combined polypeptide epitope vaccine prepared by using genetic engineering technology. Background technique [0002] HER family is a kind of epidermal growth factor receptor (EGFR) family, and its family members include: EGFR (HER1 or ErbB1), HER2 (neu or ErbB2), HER3 (ErbB3) and HER4 (ErbB4). The HER family is generally expressed in epidermal cells and stromal cells of the human body, and can regulate the proliferation and differentiation of normal cells, and the activation of its abnormal kinase activity can lead to the occurrence and proliferation of tumor cells, angiogenesis and metastasis, etc., therefore, in the mammary gland Cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, prostate cancer and other epithelial tumor cells, the overexpression of this family and its mutants can be detected. In view of this, ...

Claims

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Application Information

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IPC IPC(8): A61K39/00C12N15/62C07K19/00A61P35/00
Inventor 胡春萍曹鹏卢悟广蔡雪婷王志刚季夏芸胡云龙
Owner JIANGSU PROVINCE INST OF TRADITIONAL CHINESE MEDICINE
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