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Non-fusion expression vector capable of enhancing target gene expression and method for preparing non-fusion expression vector

A non-fusion expression and gene expression technology, applied in the field of bioengineering, can solve problems such as improvement

Active Publication Date: 2017-01-11
广东格烯生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the dual-gene expression vector, the soluble expression of the protein at the multi-cloning site 2 in E. coli is greatly improved by introducing GST into the multi-cloning site 1, and this method has not been reported so far.

Method used

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  • Non-fusion expression vector capable of enhancing target gene expression and method for preparing non-fusion expression vector
  • Non-fusion expression vector capable of enhancing target gene expression and method for preparing non-fusion expression vector
  • Non-fusion expression vector capable of enhancing target gene expression and method for preparing non-fusion expression vector

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0032] Example 1 The specific steps for the preparation of GST-containing non-fusion recombinant plasmid pETDuet-GST-CTB / EGF-CTA2-TAT are as follows:

[0033] (1) The plasmid PGEX-4T3 obtained by extraction with a plasmid extraction kit is used as a template, wherein the PGEX-4T3 has the gene sequence of GST. The BamHI-CTB-XhoI fragment was obtained by designing primers and PCR, and the PGEX-INSITE and BamHI-CTB-XhoI were digested by BamHI / XhoI at the same time to obtain the vector fragment and PCR fragment. Ligated overnight, and transformed the ligated product into E. coli BL21(DE3) competent, selected positive clones, and sent them to Shanghai Bioengineering Company for sequencing. The sequencing results showed that CTB was correctly connected to the vector PGEX, and a new plasmid PGEX-CTB was created.

[0034] (2) Using PGEX-CTB as a template, the primer sequences are as follows:

[0035] 5'-CATGCCATGGGTATGTCCCCTATACTAGGTTAT-3' (upstream primer)

[0036] 5'-CCCAAGCTTTTAA...

Embodiment 2

[0043] Example 2 Comparative analysis of EGFP-CTA2-TAT protein expression:

[0044] Transform the plasmids pETduet-GST-CTB / EGFP-CTA2-TAT and pET22b-EGFP-CTA2-TAT into competent cells BL21(DE3) respectively, and select positive transformants by ampicillin-resistant LB plate, and save as glycerol bacteria. Then, the bacterial solution was added into 400mL LB medium containing 100μg / mL at a ratio of 1:100. After culturing for 2h, 1mmol / L IPTG was added to induce expression for 6h, and the protein expression was detected by SDS PAGE and Western Blot (such as figure 2 and 3 shown).

[0045] After SDS PAGE and western blot analysis, it was found that the non-fusion expression of EGFP-CTA2-TAT of the present invention was 20 times higher than the normal expression.

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Abstract

The invention discloses a non-fusion expression vector capable of enhancing target gene expression and a method for preparing the non-fusion expression vector, and belongs to the technical field of bioengineering. Plasmids pETDuet-1 are used as skeleton vectors of the non-fusion expression vector capable of enhancing target gene expression, amino acid sequences shown as SEQ ID NO.1 are inserted into cloning sites 1 of the plasmids pETduet-1, and target genes are inserted into cloning sites 2 of the plasmids pETduet-1. The non-fusion expression vector and the method have the advantage that non-fusion expression EGFP-CTA2-TAT is higher than normal expression by 20 times as discovered.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a non-fusion expression vector capable of enhancing the expression of a target gene and a preparation method thereof. Background technique [0002] Glutathione S-transferase (GST) can improve the solubility of the target protein by fusing it with the target protein. This method is very mature. However, in the dual-gene expression vector, the soluble expression of the protein at the multiple cloning site 2 in E. coli is greatly improved by introducing GST into the multiple cloning site 1, and this method has not been reported so far. Contents of the invention [0003] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a non-fusion expression vector that can enhance the expression of the target gene. The invention provides a novel and efficient prokaryotic expression system for re...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/67
CPCC12N15/67C12N15/70C12N2800/101
Inventor 王华倩何华锋林丹敏林卫萍王甜甜潘春杏杜志云赵肃清张焜
Owner 广东格烯生物科技股份有限公司