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Preparation and application of targeted anti-tumor fusion protein lpo

A fusion protein and plasmid technology, applied in the direction of anti-tumor drugs, hybrid peptides, drug combinations, etc., can solve the problem of low tumor suppression efficiency

Inactive Publication Date: 2016-07-06
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Simple LHRH analogs also have a certain inhibitory effect, but the tumor inhibitory efficiency is relatively low

Method used

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  • Preparation and application of targeted anti-tumor fusion protein lpo
  • Preparation and application of targeted anti-tumor fusion protein lpo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: the preparation of LPO expression vector and engineering bacterium

[0054] Construction and Identification of Recombinant Expression Plasmids

[0055] a. Gene synthesis: In the present invention, according to the above-mentioned design concept, the design of the restriction site and the whole gene sequence of LPO is carried out, and the following sequence is artificially synthesized in vitro:

[0056] NcoI endonuclease recognition sequence (CCATGG)+GC+LHRH polypeptide gene+PEA transmembrane peptide+ONC sequence+stop codon (TAA)+EcoRI endonuclease recognition sequence (GAATTC), see SEQ ID NO: 1 for the gene sequence, this synthesis The sequence was directly cloned into the T vector provided by Dalian Takara Company, and transformed into Escherichia coli JM109 bacteria. The positive clones were identified by PCR, and the positive clones were expanded and cultivated. The plasmid DNA was extracted by conventional molecular cloning techniques, and digested wit...

Embodiment 2

[0059] Example 2: Expression of LPO fusion protein and purification of product:

[0060] 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 medium, and add buffer components to the bacteria containing the target protein, the final concentration reaches 50mM Tris-HCl, pH8. The precipitate (insoluble part) is the crude extract of the fusion protein.

[0061] The crude extract was washed, denatured and refolded, and the obtained refolded protein was purified by ion exchange chromatography: a DEAE-Sepharose FastFlow column (Pharmacia) equilibrate...

Embodiment 3

[0062] Example 3: Determination of the biological activity of the fusion protein using an in vitro cultured tumor cell inhibition method (for specific operations, refer to Appendix XC of the third part of the Chinese Pharmacopoeia 2010 edition).

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Abstract

The invention discloses targeting antineoplastic fusion protein LPO. An artificially synthesized target gene segment LHRH-PEA wiped membrane peptide-ONC (LPO for short) is inserted in PET28a plasmids, and the fusion protein is expressed after the LPO inserted the plasmids are conveyed in escherichia coli BL21(DE3). Experiments prove that the fusion protein LPO has obvious inhibiting effect to tumor cell lines with LHRH receptors such as colon cancer LOVO cells, lung cancer A549 cells, oral cancer KB cells and prostatic cancer PC-3M cells rather than lymphoma Hut 102 and leukemia HL60 without LHRH receptors, and has targeting specificity, and is safe and reliable.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular to targeted anti-tumor fusion protein LPO and its preparation method and application. Background technique [0002] With the development of society and the advancement of science and technology, human beings have gradually got rid of the troubles of common infectious diseases, and malignant tumors (cancers) have 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 worldwide, and this number is expected to increase to 13.2 million in 2030. According to an authoritative report issued by the National Health and Family Planning Commission, my country’s new cancers ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/70C07K19/00A61P35/00
Inventor 张国利史飞李泽鸿田园刘雨玲岳玉环吴广谋
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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