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

A technology of transgenic vector and promoter, which 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, which can solve the problem of insufficient detection sensitivity and efficiency of vector promoter. lower problem

Inactive Publication Date: 2011-04-06
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, and the detection sensitivity of some compounds is not high enough

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) Digest the plasmid rDNA-GFP with Not I at 37°C for 10 hours. The digestion system is: 5 μl 10×Buffer, 10 μg plasmid rDNA-GFP, 5 μl Not I enzyme, and add sterile water to 50 μl. The digested product is electrophoresed with 1.0% (mass ratio) agarose gel, after electrophoresis separation, the band of interest is cut off with a blade under ultraviolet light, and then the 2.1kb DNA small fragment is reclaimed with the Biospin gel recovery kit (recovery process Refer to the instructions of the Biospin Gel Extraction Kit) to obtain the HSP70-15'UTR-GFP-HSP70-1 3'UTR fragment. The recovered 2.1kb DNA fragment was then digested with BglII at 37°C for 3 hours. The digestion system was: 5 μl 10×Buffer, 30 μl recovered HSP70-15′UTR-GFP-HSP70-1 3′UTR fragment, 5 μl BglII enzyme , make up to 50 μl with sterile water. The digested product is electrophoresed with 1.2% (mass ratio) agarose gel. After electrophoresis separation, the target band ...

Embodiment 2

[0105] Plasmid T-HSP702 construction:

[0106] (1) According to Tetrahymena Genome Database (Tetrahymena Genome Database, http: / / www.ciliate.org / ) provided the whole genome sequence, and used the software Primer Premier 5.0 to design primers for the HSP70-2 gene promoter. 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 restriction site of BamH I.

[0107] (2) Using Tetrahymena thermophila SB210 genomic DNA as a template, the HSP70-2 gene promoter sequence with a length of 1132 bp was amplified with an Eppendorf PCR instrument.

[0108] (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 addition proc...

Embodiment 3

[0118] Plasmid HSP702-GFP construction:

[0119] (1) The plasmid T-HSP702 in Example 2 was digested by BamH I+Sph I at 37° C. for 2 hours, and the digested product was electrophoresed with 1.2% (mass ratio) agarose gel. Cut off the target band with a razor blade under the lamp, and then recover the 4.1kb DNA fragment with the Biospin gel recovery kit.

[0120] (2) The plasmid T-GFP in Example 1 was digested by BamH I+Sph I at 37° C. for 2 hours, and the digested product was electrophoresed with 1.2% (mass ratio) agarose gel. Cut off the target band with a razor blade under the lamp, and then use the Biospin gel recovery kit to recover a small 1.2kb DNA fragment.

[0121] (3) The 4.1 kb large fragment recovered in step (1) was mixed with the 1.2 kb small fragment recovered in step (2) at a ratio of 1:5, and T4 ligase was added to ligate overnight at 16°C.

[0122] (4) The ligation product was transformed into Escherichia coli DH5α competent, and cultured on LB solid medium co...

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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 methodssuch 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 asto 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 biological technology, and in particular relates to a Tetrahymena transgene carrier containing HSP70 promoter and GFP, and also relates to a preparation method of Tetrahymena transgene carrier containing HSP70 promoter and GFP, and also relates to containing HSP70 promoter and GFP Application of GFP Tetrahymena transgene carrier in rapid and high-throughput detection of tributyltin. technical background [0002] Heat shock protein (heat shock protein, HSP) is an important media 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 rapidly synthesize HSP as a molecular chaperone to participate in proteins The processes of synthesis, folding, assembly, operation and degradation can maintain cell protein homeostasis, improve cell tolerance to stressors, enhance anti-oxidation, and maintain normal physiolog...

Claims

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Application Information

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