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Recombinant expression vector capable of promoting protein soluble expression and increasing expression quantity

An expression vector and soluble technology, applied in the field of genetic engineering, can solve problems such as large differences in protein structure, and achieve the effects of improving protein solubility, increasing protein expression, good practical value and promotion and application significance.

Inactive Publication Date: 2017-06-13
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] But in general, due to the large differences in protein structure, when targeting different proteins, it is necessary to select a suitable tagged protein to better improve its expression level and soluble expression effect. Therefore, it is still necessary to find and design new tagged proteins or recombinant vectors , so as to meet the needs of different protein expression

Method used

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  • Recombinant expression vector capable of promoting protein soluble expression and increasing expression quantity
  • Recombinant expression vector capable of promoting protein soluble expression and increasing expression quantity
  • Recombinant expression vector capable of promoting protein soluble expression and increasing expression quantity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The recombinant expression vector pET21b-HEMBP (Pyr) that can promote protein soluble expression and increase protein expression, the construction process is as follows figure 1 As shown in (A), it is constructed through the following steps.

[0075] (1) Construction of recombinant plasmid pUC57-HEMBP(Pyr)-Nb

[0076] The artificially synthesized HEMBP(Pyr)-Nb gene sequence, the specific sequence is shown in SEQ ID NO.1, was connected with the pUC57 plasmid to construct the recombinant plasmid pUC57-HEMBP(Pyr)-Nb.

[0077] (2) Enzyme digestion and connection, the specific process is:

[0078] Plasmid pUC57-HEMBP(Pyr)-Nb and plasmid pET21b were digested with NdeI and XhoI respectively, and the 30 μL digestion system was designed as follows:

[0079] NdeI, 1 μL;

[0080] XhoI, 1 μL;

[0081] 10 × FastDigest Green Buffer, 3 μL;

[0082] Plasmid pUC57-HEMBP(Pyr)-Nb (or plasmid pET21b), 1 μg;

[0083] wxya 2 O was added to 30 μL;

[0084] Enzyme digestion at 37°C for...

Embodiment 2

[0099] On the basis of Example 1, in order to further test and determine the promotion effect of the constructed recombinant expression vector pET21b-HEMBP (Pyr) on protein expression and protein solubility, using the prokaryotic expression system, the inventors used different exogenous proteins The expression experiment was carried out, and the relevant experimental process is briefly introduced as follows.

[0100] (1) Construction of a recombinant expression vector containing the target gene

[0101] Specific target genes include H5HA10, PCV2b, NLSFMD, HAFnt, refer to figure 1 (B) Schematic diagram of the construction process, recombinant plasmid expression vectors pET21b-HEMBP(Pyr)-H5HA10, pET21b-HEMBP(Pyr)-PCV2b, pET21b-HEMBP(Pyr)-NLSFMD, pET21b-HEMBP(Pyr)- HAFnt, the specific construction process is as follows:

[0102] The recombinant expression vectors pET21b-HEMBP(Pyr)-Nb and pET21b-His6-cherry-H5HA10, pET21b-LSL150-cherry-PCV2b, pET21b-LSL150-cherry-NLSFMD, pET21b ...

Embodiment 3

[0129] Based on the recombinant expression vector constructed in Example 1, the inventor further constructed recombinant expression vectors for 10 different target genes and carried out prokaryotic expression. The 10 target genes specifically include: Nb, PCV2VHHC3, H5HA10, PCV2b , HAFnt, FMDV98, Cago60, CdiGMP499 (Caulobacter vibrioides), CdiGMP026 (Phenylobacterium zucineum), Prop acid OAS, related operations refer to Examples 1 and 2 and the prior art, and will not be repeated.

[0130] The flow chart of the construction of related recombinant expression plasmids is as follows: figure 2 As shown in (C), the results of enzyme digestion and identification of the constructed recombinant expression plasmid are as follows: Figure 4 shown. The results showed that the recombinant plasmid was constructed successfully.

[0131] In order to further demonstrate the technical effect of the present invention, based on the existing pET21b-His6MBP plasmid, referring to the relevant op...

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Abstract

The invention belongs to the technical field of gene engineering and particularly relates to a recombinant expression vector capable of promoting protein soluble expression and increasing protein expression quantity. The recombinant expression vector pET21b-HEMBP(Pyr) is a prokaryotic expression vector; after double digestion based on pET21b, a HE-MBP(Pyr)-TEV sequence is connected through recombination; and the recombinant expression vector is obtained specifically by the steps of construction of recombinant plasmid pUC57-HEMBP(Pyr)-Nb, digestion, connection, conversion, screening and the like. The recombinant expression vector can be applied to protein expression to express multiple proteins such as monoclonal antibody, antigen, polyprotein, c-di-GMP and c-GAMP synthetase. In the invention, the protein expression quantity and protein solubility can be improved relatively well by constructing and transforming to obtain a new recombinant expression vector; and the invention is of relatively good practical value and significance in popularization and application in gene engineering and protein engineering.

Description

technical field [0001] The application belongs to the technical field of genetic engineering, and in particular relates to a recombinant expression vector capable of promoting protein soluble expression and increasing protein expression. Background technique [0002] With the advancement of scientific research in the fields of genomics, proteomics and bioinformatics, recombinant DNA technology has been widely used in the application research of various target proteins, such as biochemistry, structural biology, biotechnology and other applied research. As the product of recombinant DNA technology, fusion protein is already a new class of biomolecules with multiple functional properties. The introduction of DNA recombination technology has realized the identification, transformation, expression, isolation and purification of target proteins in various host biological systems. Currently, the host biological systems that are widely used include: bacteria (E. coli), yeast, plants...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66
CPCC12N15/62C12N15/66C12N15/70
Inventor 杨国宇韩莹倩王月影李和平王江郭豫杰郭婉莹苏冰倩褚贝贝
Owner HENAN AGRICULTURAL UNIVERSITY
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