Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Diapause hormone fusion gene, fusion protein, preparation method and application

A technology of diapause hormone and fusion gene, which is applied in the field of diapause hormone fusion gene, diapause hormone fusion protein and the preparation of the fusion protein, and can solve the problems of pest physiological interference, etc.

Inactive Publication Date: 2015-01-21
HENAN UNIV OF SCI & TECH
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As one of insect endocrine-regulating neuropeptides, diapause hormone requires an extremely low dose to cause insect physiological responses. In theory, only a very small amount of exogenous DH enters the insect body to cause strong physiological disturbance to pests, but how effective is it? Maintain exogenous DH activity, remains to be resolved

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
  • Diapause hormone fusion gene, fusion protein, preparation method and application
  • Diapause hormone fusion gene, fusion protein, preparation method and application
  • Diapause hormone fusion gene, fusion protein, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Construction of recombinant plasmid pGEX-4T-TCDH:

[0046] 1. Primer design and PCR amplification

[0047] Restriction nucleases EcoRI, SalI, NotI, ordinary Taq enzyme, and T4 ligase are all products of MBI Fermentas in the United States; plasmid extraction kits, gel recovery kits, and PCR product recovery kits are products of Shanghai Sangong; other reagents used Analytical grades are BBI products.

[0048] The TCDH gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd. (the connection between TAT and Cloan-DH is based on covalent connection), and the connection was preserved in the pUC57 plasmid to form the intermediate vector pUC57-TCDH (the small linear fragment of the TCDH gene was synthesized artificially, Small fragments of double-stranded DNA are not as stable as granulated DNA. They are synthesized by the above-mentioned companies and then ligated and stored in cloning vectors. pUC57 is a commonly used plasmid vector). Primer pair A uses the pUC57-TCD...

Embodiment 2

[0097] Construction of recombinant plasmid pGEX-4T-TCDH-EGFP:

[0098] 1. Double digestion of target gene (EGFP) and vector pGEX-4T-TCDH

[0099] The PCR product (EGFP) and the vector pGEX-4T-TCDH were digested with SalI and NotI, respectively, at 37°C for 3h. Establish the enzyme digestion reaction system shown in Table 5 below in a 200 μl centrifuge tube.

[0100] Table 5 SalI, NotI double enzyme digestion reaction system

[0101]

[0102] The target gene and pGEX-4T-TCDH fragment obtained by double enzyme digestion were recovered by PCR product recovery kit for connection.

[0103] 2. Connection conversion

[0104] Ligate the obtained fragment of interest to the carrier, add reagents to the reaction system shown in Table 6 in a 500 μL centrifuge tube, and transform after 3 hours at 22°C.

[0105] Table 6 The ligation reaction system of target fragment EGFP and pGEX-4T-TCDH vector

[0106]

[0107] Transformation was carried out after 3 hours at 22°C. The transfor...

Embodiment 3

[0111] Prokaryotic expression of TCDH-EGFP fusion protein:

[0112] 1. Conversion

[0113] Rosetta (DE3) competent cell production method is the same as above (CaCl 2 Prepare fresh TOP10 Escherichia coli competent cells), and the transformation method is the same as above (TOP10 Escherichia coli competent cell transformation step).

[0114] Transform the expression strain Rosetta (DE3) with the correctly identified recombinant plasmid to obtain a single colony plate.

[0115] 2. Cultivate

[0116] (1) Pick a single colony and culture it in a test tube (5mL LB medium, 50ug / mL ampicillin, 35ug / mL chloramphenicol) overnight at 37°C and 220rpm / min;

[0117] (2) Inoculate the cultured bacteria liquid into 200ml LB medium at a ratio of 1:100, add 50ug / mL ampicillin, 35ug / mL chloramphenicol, expand the culture at 37°C and 180rpm / min;

[0118] (3) When the OD value reaches about 0.6, add IPTG with a final concentration of 0.6mM, induce at 30°C, 100rpm / min for 3.5h, and collect sam...

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

The invention discloses a diapause hormone fusion gene, a fusion protein, a preparation method and an application, and belongs to the technical field of biology. On basis of clostera anastomosis diapause hormone gene (Cloan-DH) coded sequence, a synthetic genetic code is designed and changed to obtain optimized artificial genes; with enhanced green fluorescent protein (EGFP) as a label, a TAT protein transduction domain is synthesized artificially; and the objects of preventing and controlling insects are achieved by constructing TAT-Cloan-DH (TCDH) fusion gene and expressing the fusion protein in escherichia coli, by virtue of TAT efficient transmembrane transduction of TAT.

Description

technical field [0001] The invention specifically relates to a diapause hormone fusion gene, and also relates to a diapause hormone fusion protein and a preparation method and application of the fusion protein, belonging to the field of biotechnology. Background technique [0002] TAT (transactivator of transcription, TAT) is a polypeptide sequence necessary for effective transcription and replication of viruses. The TAT protein transduction domain can non-specifically transduce the covalently linked biomacromolecules (such as nucleic acids, polypeptides, proteins, etc.) irrelevant and virtually non-toxic to host cells. Such specific polypeptide sequences capable of carrying foreign proteins to penetrate the cell membrane are called protein transduction domains (Protein transduction Domains, PTDs), also known as cell penetrating peptides (CPP). [0003] Insect diapause hormone (DH) is a kind of insect neuropeptide hormone, which controls diapause behavior. Diapause behavi...

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
IPC IPC(8): C12N15/62C12N15/70C07K19/00A01N57/16A01N47/44A01P7/04
Inventor 周洲李永丽源春彦王盼盼李红英
Owner HENAN UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com