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Construction technique and application method of diatoma tenue capable of expressing shell surface anchoring protein

An anchoring protein and construction method technology, applied in the field of genetic transformation of genes, can solve the problems of reduced biological activity of proteins, insufficient exposure of active sites, cumbersome operation steps, etc.

Inactive Publication Date: 2020-03-24
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the physical adsorption method will make the proteins randomly arrange on the surface of the material, and cannot precisely control the adsorption amount and conformation of the protein, nor can it selectively adsorb specific proteins. Reduced biological activity
The operation process of chemical adsorption method is complicated, and the addition of chemical reagents in the adsorption process may easily cause protein denaturation and inactivation, so it cannot be used on a large scale
Bioaffinity adsorption can selectively adsorb specific proteins and maintain protein activity, but the operation steps are cumbersome and the production cost is high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0007] 1. Plasmid preparation: construct the basic plasmid pLJ01 with reference to Sequence 1, design a functional gene expression sequence suitable for diatom expression according to the functional gene sequence and diatom codon preference, and insert the diatom-specific promoter and corresponding resistance selection marker Genes and specific terminators were inserted into the multiple cloning site-1 (sequence 2) of pLJ01 in sequence, and diatom-specific promoters, anchor protein gene sequences, functional gene expression sequences, fluorescent screening marker genes and specific terminators were inserted into Sequentially inserted into the multiple cloning site-2 (sequence 3) position of pLJ01 to obtain the diatom frustule surface anchor protein expression vector;

[0008] 2. Preparation of algae cells: fast-growing and easy-to-cultivate diatoms were selected as recipient algae, and a specific medium for algae was used. Seawater was taken from the sea area near Qingdao, filt...

Embodiment 1

[0013] 1. Thalassiosira pseudonana ( Thalassiosira pseudonana ) Construction of shell surface anchored lysozyme expression vector:

[0014] (1) Construction of the basic plasmid: construct the basic plasmid pLJ01 with reference to Sequence 1;

[0015] (2) Construction of diatom frustule surface anchor protein expression vector: According to the codon preference of Thalassiosira pseudonana, the lysozyme gene sequence (sequence 4) suitable for the expression of Thalassiosira pseudonana was designed, with a total gene length of 435 bp. The diatom-specific promoter Tpfcp8 promoter (sequence 5), the corresponding resistance screening marker gene Nourseothricin N-acetyl-transferase gene (sequence 6) and the unique terminator Tpfcp8 terminator (sequence 7) were sequentially inserted into the multiple cloning site of pLJ01 At -1 position; the diatom-specific promoter Tpfcp8 promoter, lysozyme gene expression sequence, anchor protein gene sequence (sequence 8), fluorescent screening ...

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Abstract

The invention discloses a construction technique and application method of diatoma tenue capable of expressing shell surface anchoring protein, and belongs to the technical field of gene heredity andtransformation. The construction technique comprises the following steps of (1) constructing basic plasmid; (2) designing a functional protein gene expression sequence; (3) constructing a diatoma tenue shell surface anchoring protein expression vector; (4) performing heredity and transformation of the diatoma tenue shell surface anchoring protein expression vector on the diatoma tenue; and (5) screening and identifying recombinant diatoma tenue so as to finally obtain transgenic diatoma tenue capable of expressing the shell surface anchoring protein. The transgenic diatoma tenue produced by the method disclosed by the invention has the capacity for efficiently expressing the shell surface anchoring protein, can be stably inherited, and can replace shifted functional genes according to practical application requirements, and the expressed shell surface anchoring protein has the activity of functional protein, and can be applied to the fields of development of animal feeds, biological fertilizer, biological pharmacy, functional foods, health-care products and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic transformation of genes, and in particular relates to a construction technology and an application method of diatoms expressing shell surface anchoring proteins. Background technique [0002] Protein is a substance with a certain spatial structure formed by the polypeptide chain composed of amino acids in the form of "dehydration condensation" after coiling and folding. It has various physiological and biochemical functions such as structural composition, catalysis, regulation, storage and transport. Protein immobilization refers to the process of using physical, chemical or biological methods to immobilize proteins on the surface of materials and maintain their activity. Immobilization of proteins on the surface of materials has a wide range of applications, such as protein analysis, drug screening, and biocatalysis. The immobilization of proteins is a complex dynamic process involving various f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N1/13C12N9/36C12Q1/6895G01N33/573C12R1/89
CPCC12N15/8216C12N9/2462C12Y302/01017C12Q1/6895G01N33/573C12N2800/22C12Q2600/158G01N2333/936
Inventor 冯超李静陈西广张凯超
Owner OCEAN UNIV OF CHINA