Construction method for domestic silkworm silk glandulae biological factory and pharmacy use

A silk and biological technology, which is applied in the fields of genetic engineering and polypeptide drug preparation, can solve the problems of unreported screening methods and achieve the effects of low cost, high biological safety and improved expression level

Inactive Publication Date: 2008-09-24
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, Invitrogen has developed a vector pIZT / V5-His that can stably express exogenous genes in insect sf cells. The vector can obtain cell lines stably expressing exogenous genes through Zeocin resistance marker selection, and Zeocin antibiotics There is no report on the screening method of transgenic silkworm

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Example 1: Preparation of transgenic silkworm in which sericin gene promoter-1 controls human insulin-like growth factor-I gene

[0034] The technical operation process is as follows:

[0035] 1. Preparation of human insulin-like growth factor-I gene

[0036] According to the cDNA sequence (GenBank accession number M11568) of the published human insulin-like growth factor-I gene, the DNA sequence corresponding to the human insulin-like growth factor-I active peptide is artificially synthesized according to the routine:

[0037] TGGATATCATGggaccggagacgctctgcggggctgagctggtggatgctcttcagttcgtgtgtggagacaggggcttttatttcaacaagcccacagggtatggctccagcagtcggagggcgcctcagacaggcatcgtggatgagtgctgcttccggagctgtgatctaaggaggctggagatggagatgtattgcgcTCacctTACT

[0038] In order to facilitate cloning and expression, a start codon ATG and a restriction site for EcoR V were introduced at the 5' end, and a stop codon TAA and a restriction site for Xho I were introduced at the 3' end.

[0039] 2. ...

Embodiment 2

[0059] Example 2: Preparation of transgenic silkworm with silk protein gene P25 promoter controlling human insulin-like growth factor-I gene

[0060] The specific embodiment is basically the same as Example one, and the difference is that step 3 in Example one is changed to "the construction of the p25 gene promoter controls the IGF-1 gene", and the specific steps are:

[0061] 1. Same as step 1 in Example 1

[0062] 2. Same as step 2 in Example 1

[0063] 3.p25 gene promoter controls the construction of IGF-I gene

[0064] About 470bp was obtained by conventional PCR amplification using the genomic DNA of the silkworm pine pine variety as a template, TPP25-1 (gcg aat tca aca gaaatc ccg ag) and TPP25-2 (ccg ata tcc gcg cca gga tgt tgc gcg) as primers The fragment of the p25 gene promoter was confirmed by sequence determination; the PCR product was digested by EcoR I and EcoR V, and then cloned into the pSK-IGF vector with the same double enzymes to obtain the recombinant pSK-P...

Embodiment 3

[0074] Example 3: Preparation of transgenic silkworm with BmNPV hr3 enhancer and silk fibroin light chain gene (Fib-L) promoter controlling human granulocyte-macrophage colony-stimulating factor gene (hGM-CSF)

[0075] The specific embodiment is basically the same as Example one and Example two, and the difference is that the human insulin-like growth factor-I gene in Example one and Example two is changed into human granulocyte-macrophage colony-stimulating factor gene, and start The promoter was changed to the silk fibroin light chain gene (Fib-L) promoter, and the enhancer was changed to the hr3 enhancer of the silkworm nuclear polyhedrosis virus (BmNPV) homologous region sequence (homologous region).

[0076] The specific steps are:

[0077] 1. Preparation of human granulocyte-macrophage colony-stimulating factor gene

[0078] According to the published cDNA sequence of human granulocyte-macrophage colony-stimulating factor gene (hGM-CSF) (GenBank accession number M11220)...

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PUM

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Abstract

The invention discloses a method for building a domestic silkworm silk gland biologic plant. A recombinant transgene vector which is provided with domestic silkworm silk protein specificity promoter controlled extraneous gene expression cassettes, an enhancer activity component, antibiotic resistant genes and fluorescent protein reporter genes and based on a piggyBAC transposon is established through gene engineering operations. The transgene vector is introduced into primarily laid eggs, special antibiotics are fed to hatched newly exuviated silkworms continuously until the fourth instar. In this way, silkworms which can resist the specific antibiotics and are provided with fluorescent protein reporter genes are obtained; target genes are detected; and transgene silkworms are manufactured by breeding. By adopting the silk gland tissues of the transgene silkworms, protein drugs can be obtained. With the method disclosed by the invention, transgene domestic silkworm silk gland plants can prepare protein drugs at low manufacture cost without potentially hazardous rhabdovirus. The method is safe and is practically valuable.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for establishing a silk gland factory of silkworm and its application in the field of polypeptide drug preparation. Background technique [0002] Achievements in the fields of biology and molecular biology research have contributed to the flourishing development of transgenic animal bioreactors. The protein produced by transgenic animals is post-processed to resemble the structure of human natural proteins, and has completely similar biological activities. The use of transgenic animal bioreactors to produce pharmaceutical proteins is another revolution in the field of biotechnology. It uses a new method to produce precious drugs. The pattern of protein used is different from the production of traditional medicines. [0003] A technical route for the production of medicinal proteins in transgenic animal bioreactors is to construct the target gene into a carri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/16C12N15/27A01K67/04A61K35/64
Inventor 贡成良曹广力薛仁宇沈卫德
Owner SUZHOU UNIV
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