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Conjugate, and preparation and use thereof

A technology of conjugates and coupling agents, applied in the field of conjugates and their preparation, can solve problems such as easy drug resistance and controversial treatment effects of patients, and achieve prolonged survival, less toxic and side effects, and high safety Effect

Active Publication Date: 2009-07-08
BIOTECH PHARMA CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these two small molecule drugs are prone to drug resistance during the treatment process, and there are disputes about the therapeutic effect on patients of different races and different living environments

Method used

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  • Conjugate, and preparation and use thereof
  • Conjugate, and preparation and use thereof
  • Conjugate, and preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1, the preparation of recombinant human epidermal growth factor mutant mixture (rhEGF)

[0066] 1. Cloning of recombinant human epidermal growth factor coding gene

[0067] According to the codon bias of yeast, the full-length DNA sequence encoding mature recombinant human epidermal growth factor was artificially synthesized, and the gene was named as its nucleotide sequence as shown in sequence 1 in the sequence listing, and the encoded amino acid sequence as shown in the sequence listing Shown in Sequence 2.

[0068] Using the full-length DNA sequence of the above-mentioned synthetic recombinant human epidermal growth factor as a template, use Primer5.0 software to design primers: upstream primer: 5-CCG CTCGAG AACTCAGATAGTGAATGCC-3, downstream primer: 5-CCG GAATTC TCAACGTAATTCCCACCA-3, in which the underlined parts are the restriction sites of XhoI and EcoRI, respectively, were amplified by PCR. The PCR reaction system is: 0.5 μl template, 5 μl 10×PCR...

Embodiment 2

[0076] Embodiment 2, the preparation of Neisseria meningitidis outer membrane protein (P64k)

[0077] 1. Cloning of the outer membrane protein coding gene of Neisseria meningitidis

[0078] According to the sequence of Neisseria meningitidis of GenBank Accession No.X77920.1, use Primer5.0 software to design primers: upstream primer: 5-CATG CCATGG CTTTAGTTGAATTGAA-3, downstream primer: 5-CCG GAATTCTTATTTTTTCTTTTGCGGAG-3, where the underlined parts are the restriction sites of NcoI and EcoRI respectively. Neisseria meningitidis CMCC29336 (purchased from China Medical Bacteria Collection Center, referred to as CMCC) was boiled at 100° C. for 10 min, and 3 μl was taken as a template for PCR amplification. The PCR reaction system is: 3 μl template, 5 μl 10×PCR buffer, 1 μl 10 mmol / L dNTP, 1 μl Pyrobest high-fidelity DNA polymerase (Shanghai Sangong product), 0.5 μl each of upstream and downstream primers with a final concentration of 0.5 μmol / L, adding Ultrapure water to 50 μl...

Embodiment 3

[0085] Example 3, Preparation of recombinant human epidermal growth factor mutant mixture and Neisseria meningitidis outer membrane protein conjugate (rhEGF-P64k)

[0086] Use 0.1M sodium borate solution (pH8.5) to adjust the concentration of the mixture of the recombinant human epidermal growth factor mutant prepared in the above example 1 and the P64k prepared in the above example 2 to 1.0 mg / mL respectively, and take different volumes of Concentration is the mixture of the recombinant human epidermal growth factor mutant of 1.0mg / mL and P64k protein solution in the aseptic pyrogen-free reaction kettle of 10L, the mol ratio that makes the mixture of recombinant human epidermal growth factor mutant and P64k is ( 12-8):1. Mix the above mixed solution in the reaction kettle and equilibrate to room temperature, slowly add 0.5% glutaraldehyde solution by volume, so that the final concentrations of glutaraldehyde in the mixed solution are 0.01%, 0.05% and 0.1% respectively . Aft...

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Abstract

The invention discloses a coupling object. The coupling object is obtained through the coupling of at least one protein in the four proteins of a recombined human epidermal growth factor and a recombined epidermal growth factor mutant with a carrier protein. The amino acid sequence of the recombined human epidermal growth factor is shown by the No.2 sequence in a sequence list. The recombined epidermal growth factor mutant can be one of the following proteins: 1)the protein obtained through the deletion of the No.53 arginine in the No.2 sequence in the sequence list; 2) the protein obtained through the deletion of the No.53 arginine and No.52 leucine in the No.2 sequence in the sequence list; and 3) the protein obtained through the addition of methionine before the No.1 asparagine in the No.2 sequence in the sequence list. The coupling object can be used as a vaccine for the active treatment of an epithelium sourced tumor after being mixed with an adjuvant. Results show that the vaccine can induct the high level antibodies in a mouse; the highest antibody titer reaches 1:20,000; and the prolonging rate of the survival period of the mouse reaches 69.4 percent.

Description

technical field [0001] The invention relates to a conjugate, a preparation method and application thereof. Background technique [0002] According to the report of the World Health Organization, there are about 10 million new cancer patients and 6 million deaths in the world every year. Since the 1970s, the incidence and mortality of malignant tumors in my country have been on the rise. In 2000, there were about 2 million cancer cases and 1.5 million deaths. At present, the death rate of cancer has surpassed that of cardiovascular and cerebrovascular diseases and has leapt to the first place. Malignant tumors have become the "first killer" of human beings. [0003] Studies have shown that epithelial tumors, including malignant tumors such as lung cancer, gastric cancer, liver cancer, esophageal cancer, colorectal cancer, cervical cancer, nasopharyngeal cancer, and breast cancer, are closely related to their occurrence, formation, proliferation, metastasis, and poor prognos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/02A61K39/385A61K47/48A61P35/00A61K47/64
Inventor 白先宏冷木李先钟喻志爱胡品良何伟林峰何丽华王艳萍艾军文
Owner BIOTECH PHARMA CO LTD
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