Method for preparing transgenic bombyx mori and its application in pharmacy

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

Inactive Publication Date: 2007-06-06
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 100% cold ethanol 2 times the volume of the supernatant, mix well, centrifuge at 12 000 rpm for 10 minutes, discard the supernatant, add conventional reverse transcriptase and 4dNTPs for reverse transcription, 37 ° C, 1 cDNA synthesized by reverse transcription of mRNA was obtained.

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

[0021] The cDNA obtained in the first step was added to the upstream and downstream primers 5'CGGAT...

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 into powder raw materials, 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, and then freeze-dry it into a refined raw material powder, and pack 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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Abstract

The present invention discloses a process for preparing human granulocyte with - intracellular method macrophage colony stimulating transgenic factor silkworm, the person under Bombyx mori nuclear polyhedrosis virus gene promoter particles by genetic engineering - macrophage colony stimulating in factor gene was cloned into the transposon-based vectors piggyBAC factor reporter gene with a recombinant gene transfer vector, plasmid role in co-axial, Bombyx mori with a reporter gene mediated by sperm, and human granulocyte-detected - macrophage colony stimulating factor gene, the transgenic silkworm breeding made.Moth larvae or pupae or using the transgenic silkworm obtained was freeze-dried green white blood drugs.The method of the present invention is to achieve a transgenic silkworm Health leukocyte pharmaceutical preparation, low production cost, the viral genome do not involve potentially harmful, safety, oral drug effects in particular good results, the embodiment of great value.

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 yea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12Q1/68A01K67/04A61K35/64
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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