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Application of recombinant Ganoderma lucidum immunoregulatory protein in the preparation of medicines for treating leukopenia caused by chemotherapy drugs

A technology for immunoregulatory proteins and leukopenia, applied in drug combinations, extracellular fluid diseases, peptide/protein components, etc.

Inactive Publication Date: 2011-12-14
张喜田
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Malignant tumors are common and frequently-occurring diseases that seriously threaten human health. At present, there is no particularly effective prevention and treatment strategy

Method used

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  • Application of recombinant Ganoderma lucidum immunoregulatory protein in the preparation of medicines for treating leukopenia caused by chemotherapy drugs
  • Application of recombinant Ganoderma lucidum immunoregulatory protein in the preparation of medicines for treating leukopenia caused by chemotherapy drugs
  • Application of recombinant Ganoderma lucidum immunoregulatory protein in the preparation of medicines for treating leukopenia caused by chemotherapy drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Obtaining of recombinant Ganoderma lucidum immunomodulatory protein

[0027] 1. Artificial synthesis of rLZ-8 gene, construction and screening of engineering bacteria

[0028] 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.

[0029] 2. Expression of rLZ-8 engineering bacteria

[0030] 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

[0038] Example 2: Killing effect of rLZ-8 on human promyelocytic leukemia cell NB4

[0039] 1. Reagents

[0040] 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 .

[0041] 2. Experimental method

[0042] 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.

[0043] 3. Experimental results

...

Embodiment 3

[0048] Example 3: Killing effect of rLZ-8 on human chronic myelogenous leukemia cell K562

[0049] 1. Reagents

[0050] 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 .

[0051] 2. Experimental method

[0052] 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.

[0053] 3. Experimental results

[0054] Table 2 and figure 2 As shown, the rLZ-8 drug...

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Abstract

The invention relates to the application of recombinant Ganoderma lucidum immunoregulatory protein rLZ-8 in the preparation of medicine for treating leukopenia caused by chemotherapeutic drugs. The invention establishes a leukopenia disease model in rats caused by cyclophosphamide, and uses the recombinant Ganoderma lucidum immunoregulatory protein to conduct treatment experiments. The experimental results prove that the recombinant Ganoderma lucidum immunomodulatory protein has a very significant effect on increasing white blood cells.

Description

[0001] This application is a divisional application of the application of "recombinant ganoderma lucidum immunoregulatory protein with anti-tumor effect and its pharmaceutical preparation". The filing date of the original application is January 3, 2008, the application number is 200810050206.x, and the name of the original invention is "recombinant Ganoderma lucidum immunoregulatory protein with anti-tumor effect and its pharmaceutical preparation". technical field [0002] 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 [0003] 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....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/16A61P7/00
Inventor 孙非刘立侠许守民梁重阳
Owner 张喜田
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