Dual-luciferase reporter gene vector for insect cells, construction method, recombinant vector and application

A dual-luciferase and reporter gene technology, applied in the field of dual-luciferase reporter gene vectors for insect cells, can solve problems such as inactivity, limited use, and inability to normally start luciferase gene expression

Pending Publication Date: 2022-05-13
XINXIANG MEDICAL UNIV
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The co-transfection experiment has the following defects: First, the length of the full-length 3'-UTR of most target genes varies from several hundred to several thousand bases, and the recombinant reporter vector is constructed
[0006] In the prior art, although there is a Psicheck-2 vector design, which can monitor changes in gene expression by fusing firefly and sea cucumber luciferase reporter genes, this vector is almost non-existent in insect cells due to the species-specificity of the promoter. Activity, can not normally start the expression of luciferase gene on the carrier, so the use in insect cells is limited

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
  • Dual-luciferase reporter gene vector for insect cells, construction method, recombinant vector and application
  • Dual-luciferase reporter gene vector for insect cells, construction method, recombinant vector and application
  • Dual-luciferase reporter gene vector for insect cells, construction method, recombinant vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 dual luciferase reporter gene carrier and its construction process

[0042] The dual-luciferase reporter gene carrier of insect gene 3'UTR in this example is obtained by modifying the psiCHECK-2 vector, specifically including replacing the original SV40 promoter with the OpIE2 promoter and inserting a synthetic DNA fragment. The synthetic DNA fragment sequence is connected between the HSV-TK promoter and Synthetic poly(A) of the psiCHECK-2 vector; the insect gene 3'UTR sequence is inserted into the hRluc downstream of the psiCHECK-2 vector; the nucleotide sequence of the synthetic DNA fragment As shown in SEQ ID NO:1. The promoter sequence of OpIE2 is shown in SEQ ID NO:2.

[0043] The above-mentioned dual luciferase reporter gene vector (pPIZ / 3UTR) was constructed with the selected vector pIZ / V5-His and vector psiCHECK-2, and the construction method is as follows:

[0044] (1) The plasmid map of vector psiCHECK-2 and pIZ / V5-His is as follows figure 1 an...

Embodiment 2

[0061] Embodiment 2 recombinant vector and application experiment

[0062] The insect cell dual luciferase reporter gene recombinant vector and application of the present embodiment are specifically described as follows:

[0063] 2.1. Construction of recombinant vector

[0064] The pPIZ / 3UTR vector was connected with the insect trehalase gene and the 3'UTR sequence of the glucose dehydrogenase gene respectively to construct recombinant plasmids pPIZ / 3UTR-Tre and pPIZ / 3UTR-GDH.

[0065] (1) 3'UTR sequence:

[0066] The 3'UTR sequence of the trehalase gene is shown in SEQ ID NO:5.

[0067] The 3'UTR sequence of the glucose dehydrogenase gene is shown in SEQ ID NO:6.

[0068] (2) Vector construction:

[0069] The construction methods of recombinant plasmids pPIZ / 3UTR-Tre and pPIZ / 3UTR-GDH were similar to those in Example 1, and the 3'UTR fragments of trehalase gene and glucose dehydrogenase gene were respectively obtained by PCR amplification. Subsequently, the above fragmen...

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 belongs to the field of reporter gene vectors, and particularly relates to a dual-luciferase reporter gene vector for insect cells, a construction method, a recombinant vector and application. The dual luciferase reporter gene vector for the insect cells is obtained by modifying a psiCHECK-2 vector; the transformation comprises the following steps: replacing an original SV40 promoter with an OpIE2 promoter, and inserting a synthesized DNA (Deoxyribose Nucleic Acid) fragment between an HSV-TK promoter and a Synthetic poly (A); the downstream of the hRluc gene of the psiCHECK-2 vector is used for inserting an insect target gene of interest; the nucleotide sequence of the synthesized DNA fragment is as shown in SEQ ID NO: 1. The dual-luciferase reporter gene vector for insect cells provided by the invention ensures powerful gene transcription and mRNA processing in insect cells, and is suitable for insect gene function research.

Description

technical field [0001] The invention belongs to the field of reporter gene carriers, and in particular relates to a dual-luciferase reporter gene carrier for insect cells, a construction method, a recombinant carrier and applications. Background technique [0002] Insects are the most numerous organisms in the world, with a large number and strong adaptability to the environment. Because of the advantages of easy access to materials, simple genetic manipulation, and obvious genetic phenotypes, it is favored by researchers and plays an important role in many aspects such as biomedicine, insecticides, and basic research. With the continuous development of molecular biology techniques, more than 800 insect cell lines have been established, which are often used in the research of immunofluorescence, protein interaction, and regulation of target genes by small molecule RNA. [0003] MiRNAs are non-coding RNA molecules of about 22 nucleotides (nt), which can degrade target mRNA o...

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): C12N15/85C12N15/53C12N15/66C12Q1/6897
CPCC12N15/85C12N15/52C12N15/66C12Q1/6897C12N2800/105C12Q2525/207
Inventor 耿少雷王天云赵小杰孙秋丽米春柳樊振林曹祥祥
Owner XINXIANG MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap