Unlock instant, AI-driven research and patent intelligence for your innovation.

Construction method using detoxiase gene as stable expression system in silkworm

A technology for stable expression and construction methods, which can be used in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., and can solve problems such as the difficulty of separation and purification, and the inability of lipase B1 to undergo glycosylation modification.

Inactive Publication Date: 2004-07-21
成都天创生物科技有限责任公司
View PDF0 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the research on detoxifying enzymes in microorganisms and resistant insects has been quite active, and has developed to the level of molecular biology. Although scholars at home and abroad have carried out gene sequencing, cloning and transgenic research on various detoxifying enzymes, especially lipase B1 The research of the lipase B1 is more detailed, and it has been successfully expressed in Escherichia coli, cyanobacteria, Gram-positive bacteria, Flavobacterium, etc., but the lipase B1 expressed by these microorganisms and algae cannot be modified by glycosylation, which hinders the separation after expression. , The purification work brings considerable difficulty
The use of silkworm as a bioreactor to synthesize economically valuable polypeptides and proteins has attracted the attention of many researchers and investors, but there has been no report on the expression of detoxification enzyme genes using silkworm as a foreign protein gene receptor

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction method using detoxiase gene as stable expression system in silkworm
  • Construction method using detoxiase gene as stable expression system in silkworm
  • Construction method using detoxiase gene as stable expression system in silkworm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Construction of "silk protein L chain-lipase B1" fusion gene expression unit

[0031] First, extract the mRNA from the posterior silk gland of the silkworm, and design the upstream and downstream primers (upstream and downstream primer sequences are: 5'-tggatccgcggcgcgttaccatatatg-3', 5'-gcgatatcacgccctgatgg according to the known sequence of the silk protein L chain gene of the silkworm. -3'), the silkworm silk protein L chain cDNA was obtained by RT-PCR amplification, about 0.8Kb, including the signal peptide sequence.

[0032] From the total DNA of silkworm, design primers based on known sequences (upstream primer sequence: 5'-tggatccgcggcgcgttaccatatatg-3', downstream primer sequence: 5'-gcgatatcacgccctgatgg-3') to amplify the 5'end of the L chain and its upstream A DNA fragment of about 1.2Kb in the promoter region, and a 0.5Kb terminator fragment at the 3'end of the L chain.

[0033] According to the lipase B1cDAN sequence reported by Mouches et al. (Proc. Natl. Acad....

Embodiment 2

[0036] Construction of integrated gene vector

[0037] The genomic DNA of the silkworm was extracted, and the A3 promoter fragment was obtained by PCR amplification according to the sequence of the kinesin gene (A3) of the cytoplasmic muscle of the silkworm. The A3 promoter fragment was connected to the GFP gene coding region sequence in an appropriate reading frame. Insert the SV40polyA signal sequence into the downstream of the fused GFP gene to form a complete GFP gene expression unit. Add a 30bp long multi-cloning site (MCS) artificial linker upstream of the complete GFP gene fragment (the nucleotide sequence is:

[0038] 5'-ACCGCGGTCTAGAGGATCCCGGGCTGCAGT-3'). The L-EB1 expression unit of the fusion gene of the lipase B1 gene and the L chain and the complete GFP gene expression unit are linked together through a multiple cloning site region to obtain a recombinant DNA fragment. Add the inverted terminal repeat sequence of the piggyBac transposon (nucleotide sequence 5'-CGCCGCG...

Embodiment 3

[0040] Helper plasmid construction

[0041] The coding region sequence of the transposase in the piggyBac transposon was cloned by PCR technology. Add the A3 promoter in front of the coding region of the transposase gene and clone it into the plasmid pUC18, which is the helper plasmid of the gene transfer system. The L-EB1 gene vector plasmid and the helper plasmid together constitute the silkworm gene transfer system. The system achieves high-frequency integration of foreign genes in the silkworm genome through transposition, with a transformation frequency of up to 7%. The system can ensure the multi-copy and high-frequency integration of the foreign target gene in the silkworm genome, and further improve the expression level of the foreign gene.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

A process for configuring the stable expression system of detoxicating gene in silkworm includes designing and synthesizing the oligodeoxyribonucleotide primer, linking its cDNA or DNA sequence to the promoter and terminator of silkworm silkprotein L chain to configure fusion gene, inserting them between two opposite repetitive terminal sequences of transposable factor piggyBac to obtain recombinant transposon, configuring transposase gene expression carrier, using said transposon and expression carrier to transfect silkworm ova, and screening and culturing the young silkworm to obtain the transgenic silkworm able to stably express the external detoxicating enzyme lipase BI.

Description

Technical field [0001] The present invention relates to the expression of detoxification enzyme genes in transgenic silkworms, and more specifically to a method for constructing a stable expression system of detoxification enzyme genes in silkworms. Background technique [0002] Pesticide poisoning and the destructive effects of pesticides on the ecological environment have attracted more and more attention from all over the world. In 1997, there were 500,000 cases of pesticide poisoning in the world alone. In 1995, there were more than 100,000 cases of pesticide poisoning in China, mainly of which were poisoned by highly toxic pesticides. In addition to acute poisoning, pesticide residues and pollution to soil, water, air, etc. are also increasing, resulting in delayed neurotoxicity and ecological balance disorders. For pesticide poisoning and pesticide pollution, atropine and pralidoxime are mainly used at present, which have certain side effects. [00...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/04C12N15/10C12N15/12C12N15/52C12N15/55C12N15/62C12N15/85C12Q1/68
Inventor 张莉李维郭聪
Owner 成都天创生物科技有限责任公司