Novel preparation technology of targeting antitumor fusion protein LPO (lipid peroxidation)

A technology for fusing proteins and gene fragments, which is applied in the field of genetic engineering and can solve problems such as low renaturation rate

Inactive Publication Date: 2014-05-21
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Abstract
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Problems solved by technology

[0029] The purpose of the present invention is to solve the problem of low renaturation rate of the targeted anti-tumor fusion protein LPO expressed and purified in the form of inclusion bodies, and to provide a new preparation process for the targeted anti-tumor fusion protein LPO

Method used

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  • Novel preparation technology of targeting antitumor fusion protein LPO (lipid peroxidation)
  • Novel preparation technology of targeting antitumor fusion protein LPO (lipid peroxidation)
  • Novel preparation technology of targeting antitumor fusion protein LPO (lipid peroxidation)

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Embodiment 1

[0056] Embodiment 1: the preparation of LPO expression vector and engineering bacterium

[0057] Construction and Identification of Recombinant Expression Plasmids

[0058] a. Gene synthesis: In the present invention, according to the above-mentioned design concept, the restriction site and the LPO whole gene sequence are designed and the following sequence is artificially synthesized in vitro:

[0059] NcoI endonuclease recognition sequence (CCATGG)+GC+LHRH polypeptide gene+PEA transmembrane peptide+ONC sequence+stop codon (TAA)+EcoRI endonuclease recognition sequence (GAATTC), the gene sequence is shown in SEQ ID NO:1, The synthetic sequence was directly cloned into the T vector provided by Dalian Takara Company, and transformed into Escherichia coli JM109 bacteria, positive clones were confirmed by PCR identification, positive clones were expanded and cultured, and plasmid DNA was extracted by conventional molecular cloning techniques, using NcoI and EcoRI double enzymes ...

Embodiment 2

[0062] Example 2: Expression of LPO fusion protein and purification of product:

[0063] Escherichia coli BL21 (DE3) (containing T 7 RNA polymerase gene) were cultured on LB agar plates containing kanamycin (50 μg / ml). After culturing, select kanamycin-resistant colonies and culture them in LB medium containing kanamycin (50 μg / ml) at 37°C. When A 600 When it reaches about 0.4~0.6, add 1mM isopropylthio-β-D-galactoside (IPTG) (final concentration 1mM), and continue culturing at 37°C for 3-4 hours to induce the expression of the target product. Then centrifuge the cells and the medium, and add buffer components to the cells containing the target protein, the final concentration reaches 50mM Tris-HCl, pH8.0, 1mM EDTA, sonicate, and centrifuge at 4°C (20,000g, 30 minutes ), and the precipitate (insoluble part) is the crude extract of the fusion protein.

[0064] The crude extract was washed, denatured and refolded, and the obtained refolded protein was purified by ion-excha...

Embodiment 3

[0065] Example 3: The biological activity of the fusion protein was determined by the tumor cell inhibition method cultured in vitro (for specific operations, refer to Appendix XC of Part III of the Chinese Pharmacopoeia 2010 Edition).

[0066] Cytopathic Inhibition Assay:

[0067] The cultured human tumor cell monolayer was digested with trypsin, the cell suspension was collected by blowing and blowing, counted by a cell counting plate, the number of cells was adjusted to 60000 / ml, and 80 μl / well was added to a 96-well culture plate (per Well 5000 cells), 5% CO 2 , and cultured at 37°C for 4h. Adjust the concentration of the prepared LPO protein sample to 1 mg / ml, the quantitative sample is filtered and sterilized, and different amounts of samples are added to each cell well according to the equal dilution method, and then the culture medium is supplemented to make the total volume 100 μl, 5% CO 2 , and cultured at 37°C for 24h. Then discard the supernatant in the cell ...

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Abstract

The invention discloses a novel preparation technology of targeting antitumor fusion protein (lipid peroxidation). The novel preparation method comprises the steps of building a fusion expression protein which takes the glutathione S-transferase A (TrxA)-His-tag (His6-Tag)-SUMO protease recognition substrate as a protein soluble expression auxiliary fragment and takes LHRH (luteinizing hormone releasing hormone)-PEA (phenylethylamine) trans-cell penetrating peptides-ONC (LPO for short) as a target segment, connecting the two proteins with each other by a correct read frame and carrier positive sequence and converting to enter into expression bacteria, finally building fusion protein which is connected with six expression substances in series, crudely extracting, carrying out metal chelating medium purification in the presence of imidazole, and carrying out SUMO protease digestion. Compared with the LPO prepared by a conventional method, the LPO prepared by the novel preparation technology disclosed by the invention can obviously improve the inhibiting effect on the tumor cell lines such as colon cancer HT-29 cells, ovarian cancer OVCAR3 cells, cervical adenocarcinoma HeLa cells and liver cancer HepG-2 cells.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a novel preparation process of targeted anti-tumor fusion protein LPO. Background technique [0002] Malignant tumor (cancer) has become the biggest killer of human life. According to the latest research data from the World Health Organization, the global cancer incidence rate will increase by 50% by 2020, that is, 15 million new cancer patients will be added every year. Not only that, the number of deaths from cancer is also rising rapidly around the world. In 2007, 7.6 million people died of cancer in the world, and this number is expected to increase to 13.2 million in 2030. According to an authoritative report issued by the Ministry of Health, the number of new cancers and cancer deaths in my country accounted for 20% and 24% of the world's total respectively. %. Therefore, the treatment of malignant tumors has become one of the important tasks of modern medicine. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70C07K19/00A61P35/00
Inventor 张国利史飞于佳何苗田园于新海刘雨玲吴广谋岳玉环
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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