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Method for preparing transgenic bombyx mori and its application in pharmacy

A transgenic and silkworm technology, applied in biochemical equipment and methods, microbe measurement/inspection, use of vectors to introduce foreign genetic material, etc., to achieve the effects of low cost, high biological safety, and simple preparation process

Inactive Publication Date: 2005-09-21
SUZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are no reports or patents of using this method to obtain the transgenic silkworm with hGM-CSF

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: Preparation of transgenic silkworm

[0017] The technical operation process is as follows:

[0018] 1. Preparation of total cellular mRNA of human T-lymphoid cell line

[0019] Add 100 mg of human T-lymphoid cell line cells, grind with liquid nitrogen, add 1 mL of Trozol RNA extraction solution produced by GIBCO BRL Company, shake gently for 10 minutes, add 500 μL of chloroform, place at room temperature for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes. Take the supernatant, add 2 times the supernatant volume of 100% cold ethanol, mix well, centrifuge at 12000rpm for 10 minutes, discard the supernatant, add conventional reverse transcriptase and 4dNTPs for reverse transcription, 37 ° C, 1 hour, Obtain cDNA synthesized by reverse transcription of mRNA.

[0020] 2. cDNA gene preparation of granulocyte-macrophage colony-stimulating factor

[0021] Add the cDNA obtained in the first step to the upstream and downstream primers 5'CGGATATCATGTGGGCTG...

Embodiment 2

[0035] Embodiment two: the breeding of transgenic silkworm with hGM-CSF gene

[0036] G 0 G 1 Generation, G 1 The moth circles of the same generation were reared separately after hatching (the same below), and the G 1 Generation of silkworm larvae, in combination with PCR detection (the 7th step of Example 1, the same below), will have both obvious fluorescence display and the same moth circle that can detect hGM-CSF gene by PCR. 1 Cross-breeding transgenic silkworms to obtain G 2 Generation, use the above from G 1 Generation to get G 2 generation method, the G 2 Breeding preparation for G 3 Generation, on G 2 , G 3 Fluorescence detection and PCR detection are also carried out on the same generation, and the G 2 Generation to G 3 Generation G without trait segregation 3 generation. to reserved G 3 The same silkworm species of the same generation were divided into two groups, one group was reserved, and the other group was raised normally, and a single-pair hybrid...

Embodiment 3

[0037] Example 3: Preparation of Oral Capsules and Bioactivity Test

[0038] Collect the larvae or silkworm chrysalis of the hGM-CSF gene-transferred silkworm species, after homogenization, filter with gauze to remove coarse impurities, freeze-dry to become powder raw material, dissolve the raw material powder at a ratio of 1 L of water per kg, and centrifuge at 18,000 rpm for 1 hour. Take the supernatant, freeze-dry it, and turn it into refined raw material powder, and put the raw material powder into capsules to make oral capsules. Oral capsules have been proved by mouse animal experiments to have obvious effect of promoting leukocyte production.

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PUM

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Abstract

This invention discloses a method for preparing trans-gene cultivated silkworm with human granulocyte, namely macrophage colony stimulating factor, characterized by the following steps: a, through gene engineering operation, cloning human granulocyte under the control of cultivated silkworm's karyotype polyhedrosis virogene promoter into a carrier with reporting gene based on piggyBAC factor; b, constructing and recombining trans-gene carrier; c, at the presence of subsidiary particles, attaining silkworm with reporting gene by spermium conducting method, and testing out human granulocyte, breeding to silkworms.

Description

technical field [0001] The invention relates to a method for preparing a transgenic silkworm in the field of genetic engineering and a method for producing polypeptide drugs by using the obtained transgenic silkworm. Background technique [0002] Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is an important hematopoietic cell growth factor, which has specific stimulation ability for the proliferation, differentiation and maturation of granulocytes and macrophages, and It also plays an important role in improving the body's immunity, anti-tumor, and controlling autoimmune diseases. It is mainly used in leukopenia caused by cancer chemotherapy, bone marrow transplantation, myelodysplastic syndrome, aplastic anemia and as an adjuvant drug for AIDS. [0003] At present, for the development and research of hGM-CSF gene medicine, Escherichia coli expression system or yeast expression system is mainly used, but the expression level of y...

Claims

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Application Information

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IPC IPC(8): A01K67/04A61K35/64C12N15/85C12Q1/68
Inventor 贡成良曹广力薛仁宇何泽魏育红朱越雄沈卫德夏庆友周泽扬向仲怀
Owner SUZHOU UNIV
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