Recombinant Ganoderma lucidum immunoregulation protein with antineoplastic function and medicinal preparation thereof

A technology for immunoregulatory proteins and pharmaceutical preparations, applied in antineoplastic drugs, medical preparations containing active ingredients, drug combinations, etc.

Inactive Publication Date: 2009-07-08
张喜田
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Malignant tumors are common and frequently-occurring diseases that seriously threate...

Method used

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  • Recombinant Ganoderma lucidum immunoregulation protein with antineoplastic function and medicinal preparation thereof
  • Recombinant Ganoderma lucidum immunoregulation protein with antineoplastic function and medicinal preparation thereof
  • Recombinant Ganoderma lucidum immunoregulation protein with antineoplastic function and medicinal preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Obtaining of recombinant Ganoderma lucidum immunomodulatory protein

[0026] 1. Artificial synthesis of rLZ-8 gene, construction and screening of engineering bacteria

[0027] According to the genetic code preference of Pichia pastoris, on the basis of the original Ganoderma lucidum immunomodulatory protein gene sequence, the rLZ-8 gene was redesigned for whole gene synthesis, and linked with the yeast α-factor leader peptide coding sequence to form a fusion gene. Cloned into pMD18-T vector. Linearize the correctly sequenced vector, transfer it into the yeast genome, and screen methanol on the MM and MD plates to utilize the high-efficiency Mut + strain.

[0028] 2. Expression of rLZ-8 engineering bacteria

[0029] The temperature, rotation speed, pH value, liquid volume, methanol addition and other conditions of large-scale fermentation expression were tested, and the process condition optimization method for yeast engineering bacteria expressing rLZ-8 in...

Embodiment 2

[0037] Example 2: Killing effect of rLZ-8 on human promyelocytic leukemia cell NB4

[0038] 1. Reagents

[0039] After rLZ-8 was sterilized, it was prepared into 8 concentrations with IMDM culture medium, each of which was 0.78 μg ml -1 , 1.56 μg·ml -1 , 3.125 μg·ml -1 , 6.25 μg·ml -1 , 12.5 μg·ml -1 , 25 μg·ml -1 , 50μg·ml -1 , 100μg·ml -1 .

[0040] 2. Experimental method

[0041] In a 96-well culture plate, add 0.1ml of NB4 tumor cells and 0.1ml of rLZ-8 to the test wells, and the concentration of rLZ-8 increases from low to high; add 0.1ml of NB4 tumor cells and culture medium to the negative control group; add arsenic trioxide As to the positive drug control group 2 o 3 ; 6 replicate holes were made in each group. Set at 37°C, 5% CO 2 48h in the incubator, add MTT15μl (5mg ml -1 ), add 100μl 0.1mol L after the termination of cell culture 1 Isopropanol hydrochloride was used to measure the OD value at 570 nm on an enzyme-linked immunosorbent detector.

[004...

Embodiment 3

[0047] Example 3: Killing effect of rLZ-8 on human chronic myelogenous leukemia cell K562

[0048] 1. Reagents

[0049] After rLZ-8 was sterilized, it was prepared into 6 concentrations with IMDM culture medium, each of which was 3.125 μg·ml -1 , 6.25 μg·ml -1 , 12.5 μg·ml -1 , 25 μg ml -1 , 50μg·ml -1 , 100μg·ml -1 .

[0050] 2. Experimental method

[0051] In a 96-well culture plate, add 0.1ml of K562 tumor cells and 0.1ml of rLZ-8 to the test wells, and the concentration of rLZ-8 increases from low to high; add 0.1ml of K562 cells and 0.1ml of culture medium in the negative control group; positive drug arsenic trioxide; multiple holes. Set at 37°C, 5% CO 2 48h in the incubator, add MTT15μl (5mg·ml -1 ), add 100μl 0.1mol L after the termination of cell culture -1 Isopropanol hydrochloride was used to measure the OD value at 570 nm on an enzyme-linked immunosorbent detector.

[0052] 3. Experimental results

[0053] Table 2 and figure 2 As shown, the rLZ-8 drug...

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Abstract

The invention discloses a recombinant Ganoderma Lucidum immunomodulatory protein (rLZ-8) which has anti-tumor effect and is expressed by Pichia pastoris, and a pharmaceutical preparation thereof. The rLZ-8 has the effect of directly killing or damaging NB4 or K562 or HL-60 or S180 or H22 cells and not affecting normal cells; and the rLZ-8 can quickly and efficiently induce a plurality of tumor cells to apoptosize in vitro, can also effectively kill the tumor cells in a body of a mouse tumor model, has no obvious toxic side effect, and can keep or raise the level of leucocytes. In addition, the invention also provides an anti-tumor pharmaceutical preparation type taking the rLZ-8 as a core composition.

Description

technical field [0001] The invention belongs to the field of biomedical engineering, and relates to the anti-tumor application and anti-tumor drug preparation of recombinant Ganoderma lucidum immunoregulatory protein expressed by Pichia pastoris. Background technique [0002] Ganoderma lucidum immunomodulatory protein (Immunoregulatory Protein of Ganoderma lucidium), the small molecule protein (Kohsuke Kino et al., J.Boil.Chem.1989, 1:472-478), named LZ-8, and determined its amino acid sequence and immunophysiological activity. Protein sequencing showed that LZ-8 is composed of 110 amino acid residues, the amino terminal is acetylated, the molecular weight is 12.4KD, and the isoelectric point is 4.4. The main function of Ganoderma lucidum immunoregulatory protein is that it can promote the proliferation of peripheral lymphocytes and spleen cells, induce animal and human macrophages to secrete various cytokines (such as interleukin, tumor necrosis factor and interferon, etc....

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/81A61K38/16A61K9/08A61K9/20A61K9/48A61P35/00A61P7/00
CPCC07K14/375A61K38/00A61P31/00A61P31/04A61P31/06A61P31/12A61P33/02A61P35/00A61P35/02A61P37/02A61P37/06A61P7/00A61P7/02A61P7/06Y02A50/30C07K14/37
Inventor 孙非刘立侠许守民梁重阳
Owner 张喜田
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