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Cold-shock expression T-vector and application method thereof

A carrier and cold shock technology, applied in the field of bioengineering, which can solve the problems of cytotoxicity, unfavorable in situ mutagenesis and screening of target genes, and large pET vector.

Inactive Publication Date: 2016-04-20
邵蔚蓝
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the expressive T vector pETG combines TA cloning technology in the cloning and expression of the target gene, since it is derived from the T7 series vector pET, the live and high-efficiency expression of most proteins will also encounter inducers, cytotoxicity, and inclusion bodies. The problem
At the same time, the pET vector is relatively large, which is not conducive to in situ mutagenesis and screening of the target gene

Method used

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  • Cold-shock expression T-vector and application method thereof
  • Cold-shock expression T-vector and application method thereof
  • Cold-shock expression T-vector and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1. Preparation of T carrier of the present invention

[0042] Materials and Methods:

[0043] Escherichia coli ( E. coli ) strains BL21 (DE3) and DH10B were used as gene cloning and expression hosts. The source or preparation of the plasmid pHsh used is referred to (Wu et al., Biotechnol Lett , 2010, 32:795–801). restriction endonuclease Bfu The design of the I restriction site refers to the method adopted by Zhong Xing et al. (Zhong Xing et al., Acta Bioengineering Sinica, 2012, 29:510-519). The electroporation system (Bio-RadGenePulserXcell?) was used for gene transformation and the electroporation of Escherichia coli was carried out according to its operating requirements. E. coli transformants were cultured in LB medium (30-37°C).

[0044] restriction endonuclease Bfu I was purchased from Fermentas; T4DNA ligase, PrimeSTAR ? HSDNA polymerase and DNA molecular weight marker were purchased from Takara Biological Company (Dalian); DNA Fragment ...

Embodiment 2

[0055] The TA cloning test of embodiment 2.PCR product:

[0056] Because the pEXC-T carrier is a mature T carrier that itself carries the initiation codon ATG; therefore, use 3' terminal transferase activity TaqDNA polymerase amplifies the target gene. The target gene to be cloned and expressed can be the open reading frame without the translation start codon, that is, starting after the start codon ATG at the 5' end until the stop codon TAA, TAG or TGA. If you want to use the histidine tag (His-Tag) at the 3' end for later purification, you also need to remove the stop codon, but you need to consider the A (adenylic acid) that is introduced at the 3' end when cloning at the 3' end. frameshift mutation). After the amplified fragment is connected with pEXC-T, transform the competent cells, spread the transformed bacteria solution on the LB plate with chloramphenicol resistance; culture at 37°C for 15-20 hours, and select the transformed cells on the plate Sons were subject...

Embodiment 3

[0065] Example 3. Optimization of Induced Expression Conditions

[0066] The gene expression plasmid pEXC-lacZ obtained from Example 2 was electrotransformed into E. coli DH10B, spread on the LB plate containing chloramphenicol, culture at 37°C, after a single colony grows on the plate, pick a single colony and transfer it to LB liquid for culture at 37°C, and then conduct the gene expression test in an instant cooling . The study of expression conditions mainly focuses on several aspects such as the optimal induction temperature for low temperature induction, the optimal induction timing and the optimal culture period after induction.

[0067] 1. Optimum Induction Temperature Test

[0068] by lacZ Optimal induction temperature for gene expression in pEXC was investigated for the reporter gene. When the medium is LB, the cell density of the recombinant cells is OD 600 = 1.8, the temperature was 10°C-25°C and other temperature conditions, and the cold shock was induce...

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Abstract

The invention provides a cold-shock expression T-vector and an application method thereof. Proteins coded by genes of animals, plants and normal temperature microbes are not stable, and most of the proteins are denaturated within a plurality of hours after heterogeneous expression; and proteins have inhibitory or poisoning effect on growth of recombinant cells, so recombinant expression is difficult to realize. The novel T-vector designed and constructed in the invention has the following characteristics: (1) the T-vector expresses an exogenous gene in a low temperature environment and can effectively prevent protein denaturation or reduce cytotoxicity; (2) the T-vector has mall plasmid molecular weight and the characteristics of great gene capacity, a high conversion rate, etc., and can improve gene cloning efficiency; (3) a plasmid copy number is high, so the level of expression is further increased; and (4) through combination with TA cloning technology and a low temperature expression function, the constructed T-vector is applicable to library construction and activity screening. As the vector provided by the invention is used for PCR cloning of coding genes of proteins with lability or cytotoxicity, construction and screening of a gene library and over-expression of the genes, operation steps can be simplified, materials and time can be saved, and the accuracy and success rate of testing can be improved.

Description

technical field [0001] The present invention belongs to the field of bioengineering, and specifically relates to a novel T vector suitable for technical fields such as molecular biotechnology, microbial engineering, and genetic engineering, and more specifically relates to the structure, properties and properties of a low-temperature expression T vector induced by cold shock. Application Technology. Background technique [0002] The technology of using genetic engineering to produce the target protein has been widely used. Among them, Escherichia coli ( Escherichiacoli ) expression system consists of two parts: expression vector and corresponding host cell, and is the earliest, most widely used, and easiest-to-operate high-efficiency gene expression system. In the E. coli expression system, expression vectors usually use regulatable promoters to control the expression of foreign genes; the types of these promoters can reduce the inhibition of the expressed target protein o...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/64
Inventor 邵蔚蓝何燕斌
Owner 邵蔚蓝
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