Tetrahymena transgenic carrier containing HSP70 promoter and GFP and preparation method and use thereof

A technology of transgenic vector and promoter is applied to the preparation of Tetrahymena transgenic vector, and the application field of Tetrahymena transgenic vector in the rapid and high-throughput detection of tributyltin can solve the problem of low efficiency of vector promoter and insufficient detection sensitivity higher question

Inactive Publication Date: 2009-11-25
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the efficiency of the constructed vector promoter is low,

Method used

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  • Tetrahymena transgenic carrier containing HSP70 promoter and GFP and preparation method and use thereof
  • Tetrahymena transgenic carrier containing HSP70 promoter and GFP and preparation method and use thereof
  • Tetrahymena transgenic carrier containing HSP70 promoter and GFP and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Plasmid T-GFP construction:

[0099] (1) The plasmid rDNA-GFP was digested with Not I for 10 hours at 37°C. The digestion system was: 5 μl 10×Buffer, 10 μg plasmid rDNA-GFP, 5 μl Not I enzyme, and filled with sterile water to 50 μl. The digested product was electrophoresed on a 1.0% (mass ratio) agarose gel. After electrophoresis was separated, the target band was cut with a blade under ultraviolet light, and then the 2.1kb DNA fragment was recovered using the Biospin gel recovery kit (recovery process) Refer to the Biospin gel recovery kit instructions) to obtain the HSP70-15'UTR-GFP-HSP70-1 3'UTR fragment. The recovered small 2.1kb DNA fragment was digested with BglII for 3 hours at 37°C. The digestion system was: 5μl 10×Buffer, 30μl recovered HSP70-15'UTR-GFP-HSP70-1 3'UTR fragment, 5μl BglII enzyme , Make up sterile water to 50μl. The digested product was electrophoresed on a 1.2% (mass ratio) agarose gel. After electrophoresis was separated, the target band was cut wit...

Embodiment 2

[0104] Construction of plasmid T-HSP702:

[0105] (1) According to Tetrahymena Genome Database (Tetrahymena Genome Database, http: / / www.ciliate.org / ) Provided the whole genome sequence, use the software Primer Premier 5.0 to design the HSP70-2 gene promoter primer. The primers used are as follows: Upstream primer: 5′- GCG GCC GC A ATT GTT CTT GCT TGT TTT GG-3′, where GCG GCC GC Not I restriction site; Downstream primer: 5′ GGA TCC AGC TTT TTA TTT TCC AGA CAT-3', where GGA TCC It is the BamH I restriction site.

[0106] (2) Using Tetrahymena thermophila SB210 genomic DNA as a template, a 1132 bp length HSP70-2 gene promoter sequence was amplified with Eppendorf PCR machine.

[0107] (3) PCR reaction system components: 5μl 10×Buffer (containing MgCl 2 ), 1μl dNTP (10mM), 1μl upstream primer (10μM), 1μl downstream primer (10μM), 1μl DNA, 0.5μl TITANIUM TM Taq DNA Polymerase, add double distilled water to 50μl. The sample loading process is operated on ice. After the addition, ...

Embodiment 3

[0117] Construction of plasmid HSP702-GFP:

[0118] (1) The plasmid T-HSP702 in Example 2 was digested with BamH I+SphI at 37°C for 2 hours. The digested product was electrophoresed on a 1.2% (mass ratio) agarose gel. Next, cut the target band with a blade, and then use Biospin gel recovery kit to recover 4.1 kb DNA fragment.

[0119] (2) The plasmid T-GFP in Example 1 was digested with BamH I+SphI at 37°C for 2 hours, and the digested product was electrophoresed on a 1.2% (mass ratio) agarose gel. Next, cut the target band with a blade, and then use Biospin gel recovery kit to recover 1.2kb DNA fragments.

[0120] (3) Mix the 4.1 kb large fragment recovered in step (1) with the 1.2 kb small fragment recovered in step (2) at a ratio of 1:5, and add T4 ligase to ligate overnight at 16°C.

[0121] (4) The ligation product was transformed into E. coli DH5α competent, and cultured on LB solid medium containing 50μg / ml ampicillin for 16 hours.

[0122] (5) Pick a single colony and tran...

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Abstract

The invention discloses a tetrahymena transgenic carrier containing HSP70 promoter and GFP and preparation method and use thereof. Plasmid T-GFP is constructed by using plasmid rDNA-GFP in the methods such as enzyme cutting and PCR enzyme cutting site mutation; thermophile tetrahymena genome DNA is used for constructing plasmid T-HSP702 in the methods such as PCR augmentation and clone; Plasmids T-GFP, T-HSP702 and pD5H8 are used for construction and finally obtaining plasmid HSP702-GFP-pD5H8 by enzyme cutting and connection. The carrier contains HSP702 promoter, thereby being capable of realizing high-efficient expression of exogenous gene; the carrier contains replaceable exogenous gene gfp so that an exogenous gene expression system is realized for tetrahymena by replacing different exogenous genes; with the sieving effect of paromomycin medicine, the carrier replaces most rDNAs originally in the tetrahymena so that the exogenous genes are largely increased in the tetrahymena so as to realize genetic transformation of target genes. The method in the invention is easy and has convenient operation, and the carrier can be used for detection of environmental tributyl tin in transfection in tetrahymena.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a Tetrahymena transgenic vector containing HSP70 promoter and GFP, and also relates to a preparation method of a Tetrahymena transgene vector containing HSP70 promoter and GFP, and also relates to a method for preparing a Tetrahymena transgene vector containing HSP70 promoter and GFP. Application of GFP-based Tetrahymena transgenic vector in rapid and high-throughput detection of tributyltin. technical background [0002] Heat shock protein (HSP) is an important mediator molecule for organisms to adapt to environmental temperature or stress changes. Too high or too low temperature will induce a stress response in cells or the body, and quickly synthesize HSP as a molecular chaperone to participate in protein The process of synthesis, folding, assembly, operation and degradation of the cell is to maintain the stability of cell protein, improve the tolerance of the cell to stress...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12Q1/02G01N21/76C12R1/90
Inventor 冯立芳缪炜
Owner INST OF AQUATIC LIFE ACAD SINICA
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