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Silkworm silk gland recombinant expression vector for expressing human epidermal growth factor as well as preparation method and application thereof

An epidermal growth factor and expression vector technology, which is applied to the field of Bombyx mori silk gland recombinant expression vector and its preparation, can solve the problems of reduced biological activity of F-POI, affecting the application promotion and inactivation of biologically active protein functional silk materials, and the like. Effects of high human epidermal growth factor biological activity, improved biological activity, and high structural similarity

Active Publication Date: 2021-05-28
SOUTHWEST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the study found that the actual molecular weight of F-POI was significantly higher than the theoretical molecular weight of the different types of F-POI created by the traditional SF-h expression system due to the influence of redundant post-translational modifications; and the F-POI Compared with natural POI, the biological activity is significantly reduced or even completely inactivated, which seriously affects the application and promotion of functional silk materials based on genetically improved bioactive proteins

Method used

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  • Silkworm silk gland recombinant expression vector for expressing human epidermal growth factor as well as preparation method and application thereof
  • Silkworm silk gland recombinant expression vector for expressing human epidermal growth factor as well as preparation method and application thereof
  • Silkworm silk gland recombinant expression vector for expressing human epidermal growth factor as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0030] Construction of Bombyx mori silk gland recombinant expression vector expressing human epidermal growth factor

[0031] Silkworm heavy chain promoter sequence (FHP3) and light chain binding site sequence ( LBS), the Bombyx mori nuclear polyhedrosis virus enhancer hr3 sequence (the nucleotide sequence is SEQ ID NO.1), the sequence optimization truncated promoter sequence encoding the 1-21 amino acids of the N-terminal domain of the SF-h protein (FHP3 s , nucleotide sequence is SEQ ID NO.2), optimized human epidermal growth factor (EGF) gene (nucleotide sequence is SEQ ID NO.3, amino acid sequence is SEQ ID NO.4, POI (EGF)) and coding SF-h protein C-terminal domain 1-20 amino acid truncated SF-h gene light chain binding site sequence (LBS s , and the nucleotide sequence is SEQ ID NO.5), all of which were synthesized by GenScript Company in Nanjing, China.

[0032] GenScript spliced ​​the above gene sequences into FHP3-EGF-LBS and hr3-FHP3 respectively in a seamless way ...

Embodiment 2

[0034] Preparation of transgenic silkworm

[0035] The commercial diapause silkworm strain 932 was used as the raw material, and the parental silkworm eggs were treated with low-temperature catalysis at 16°C to release the diapause of the offspring silkworm eggs; then the recombinant vector NEC or N s EC s Mix it with the helper plasmid pHA3PIG at a molar ratio of 1:1, then inject it into the G0 generation silkworm eggs released from diapause, seal it with non-toxic glue, and place it in an environment with 25°C and a relative humidity of 85% to accelerate the incubation; The G0 generation ant silkworms were reared with mulberry leaves until they turned into moths, and the obtained G0 generation silkworm moths were produced by backcrossing or selfing to produce seeds, and the G1 generation silkworm eggs were collected; A motorized macroscopic fluorescence microscope was used to screen the positive transgenic silkworms with red fluorescence in the eyes or nervous system from ...

Embodiment 3

[0040] Detection of F-POI(EGF) Protein Expression in Transgenic Cocoon Silk

[0041] In order to extract mixed SF and F-POI(EGF) from silkworm cocoons for further material preparation, silkworm cocoons should be cut into pieces and degummed to remove the sericin layer. The specific operation steps of the degumming treatment are as follows: NEC and N cells expressing two different F-POI (EGF) proteins were randomly selected. s EC s Silkworm cocoons were cut into small pieces of 0.5-1cm, soaked in 0.08% (w v -1 ) sodium carbonate and 0.12% (w v -1 ) in a mixture of Marseille soap (Marseille soap), degumming at 70°C for 45 minutes, and rinsing with ultrapure water of Milli-Q system (Millipore, Billerica, MA) for 3-4 times to remove sericin.

[0042] To identify two F-POI (EGF) proteins in NEC and N s EC s Distribution in silk fibers, transgenic silk fiber samples prepared for tissue immunofluorescence examination. The specific operation steps are as follows: undegummed or d...

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Abstract

The invention particularly relates to a silkworm silk gland recombinant expression vector for expressing a human epidermal growth factor as well as a preparation method and application thereof, and belongs to the field of biological materials. The constructed recombinant expression vector is formed by fusing a truncated fibroin heavy chain N-terminal structural domain, a C-terminal structural domain, an optimized human epidermal growth factor and a silkworm nuclear polyhedron virus enhancer, a redundant part of an endogenous fibroin extra structural domain contained in fusion protein is removed to the maximum extent, on the premise of not influencing the genetic hybridization process that the fusion exogenous target protein is integrated into a silk-based material, redundant post-translation modification existing in the fusion protein is eliminated as much as possible, so that the functional silk material for expressing the human epidermal growth factor with high biological activity is created by genetic hybridization; and a feasible solution is provided for developing a novel silk-based material with high biological activity by utilizing an advanced biosynthesis platform represented by silkworms.

Description

technical field [0001] The invention belongs to the field of biological materials, and in particular relates to a silkworm silk gland recombinant expression vector expressing human epidermal growth factor and its preparation method and application. Background technique [0002] Silk, also known as cocoon silk, is a continuous long fiber formed by the solidification and fibrosis of the liquid silk protein secreted by the silk gland when the silkworm spins and cocoons. Silk is a porous protein fiber, and as a textile material, it has the advantages of good air permeability and excellent hygroscopicity. In addition, silk has excellent mechanical properties, environmental stability, biocompatibility and biodegradability and other physical and chemical properties, making it gradually emerge as an ideal biomaterial in the fields of medicine and biological tissue engineering research. In recent years, silk as a raw material has gradually been widely used in the development and res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C07K14/435
CPCC12N15/85C07K14/485C07K14/43586C12N2800/22C12N2830/008
Inventor 龙定沛程岘孙鉴锋刘焱萍代方银向仲怀
Owner SOUTHWEST UNIV
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