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Artificially constructed araneus ventricosus dragline silk protein gene and construction method thereof

A technology of dragging silk protein and Edenia spp. applied in the field of genetic engineering, which can solve the problems of low spider silk production, inability to raise spider silk at high density, and difficulty in processing full-length gene sequences, so as to save costs and improve work efficiency , The process is simple and efficient

Inactive Publication Date: 2017-05-31
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, spiders are cannibalistic and cannot be raised in high density to obtain a large amount of spider silk, and the silk production of spiders is small, so it cannot meet the needs of production and application. Therefore, people hope to use the method of genetic engineering, that is, to transfer the spider silk gene into different species. To express spider proteins in organisms, because the spider silk protein gene is a huge repetitive sequence, it is very difficult to process the full-length gene sequence, so artificially constructing a polymer spider silk gene is one of the important steps in this kind of work
However, there are no relevant studies and reports on the artificial construction of the high-molecular traction silk protein gene of E.

Method used

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  • Artificially constructed araneus ventricosus dragline silk protein gene and construction method thereof
  • Artificially constructed araneus ventricosus dragline silk protein gene and construction method thereof
  • Artificially constructed araneus ventricosus dragline silk protein gene and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Preparation of Monomer Sequence of Tractor Silk Protein Gene

[0060] The flow chart is attached figure 1 shown.

[0061] 1. According to the published partial cDNA sequence of the pulley silk protein of E. grandina (JN857964.2), determine the core repeat sequence 108bp (as shown in SEQ ID NO.1) as the basic gene composition of the monomer.

[0062] Then in the above core repeat sequence 5 ’ The Nhe I restriction site GCTAGC is added to the end, and the Spe I and HindIII restriction site ACTAGTAAGCTT is added to the end. The total length of the sequence thus formed is 126bp (as shown in SEQ ID NO.2), which is the pull silk Monomer sequences of protein genes.

[0063] Two oligonucleotide single strands (72bp, sequence shown in SEQ ID NO.3) are artificially synthesized from both ends of the gene monomer, and 18 bases are complementary at the ends of the two oligonucleotide single strands.

[0064] 2. Take 100ng of the two synthesized oligonucleotide chains (...

Embodiment 2

[0065] Example 2 Construction of pSIMPLE-19 EcoR V / BAP monomer recombinant plasmid

[0066] 1. Take 5uL of the double-stranded sequence of the pulley silk protein gene monomer of the above-mentioned fill-in product, and carry out agarose gel electrophoresis, and use Takara’s TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 gel recovery kit to detect the target article Take 126bp for rubber tapping recovery.

[0067] 2. Phosphorylate the above-mentioned reclaimed leveling product:

[0068] The reaction system is:

[0069] wxya 2 O 10.25 μl,

[0070] 10×T4 DNA Polynucleotide Kinase Buffer 2.5 μl,

[0071] 12 μl of the above recovered fill-in product,

[0072] ATP 25 nmol (0.25 μl),

[0073] T4 Polynucleotide Kinase 10 units.

[0074] The reaction conditions are: react at 37° C. for 30 minutes.

[0075] 3. Then connect the phosphorylated fill-in product to the pSIMPLE-19 EcoR V / BAP expression vector:

[0076] The reaction system is:

[0077] pSIMPLE-19 EcoR V / BAP ...

Embodiment 3

[0084] Example 3 Constructing multiple concatemers of the core repeat sequence of the pulley silk protein gene

[0085] 1. Cultivate the bacterial solution transformed with the pSIMPLE-19 EcoR V / BAP haploid recombinant plasmid, and extract the plasmid using the TIANprep Mini Plasmid Kit.

[0086] The plasmid was double-digested with restriction endonuclease Nhe I / Hind III, and the reaction composition and steps were as follows:

[0087] pSIMPLE-19 1×plasmid 2μg

[0088] 10 × Quickcut buffer 5μL

[0089] NheI 1 μL

[0090] HindⅢ 1μL

[0091] dd H 2 O to make up to 50 μL.

[0092] The above reaction system was mixed gently, centrifuged briefly, and incubated at 37°C for 2h.

[0093] 2. Separate by 1% agarose gel electrophoresis and analyze the size of the fragments (see Figure 5 ), using the DNA gel extraction kit (TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0) to recover the target gene fragment; then the single recombinant plasmid was double-digested with rest...

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Abstract

The invention discloses an artificially constructed araneus ventricosus dragline silk protein gene and a construction method thereof. The sequence of the araneus ventricosus dragline silk protein gene is shown as SEQIDNO.6. According to part of cDNA sequence of araneus ventricosus dragline silk protein, first, most-representative core repeat 108BP is selected, and artificially synthesized, both ends of the core repeat sequence are additionally added restriction enzyme cutting sites, then the core repeat sequence is connected with a pSIMPLE-19EcoRV / BAP vector, and transformed into Escherichia coli to obtain monomer recombinant plasmid, by an isocaudarner recursive directional ligation method, gene monomers are polymerized into a 16-time concatemer, 1836bp araneus pseudoventricosus dragline silk protein gene can be successfully obtained, the 1836bp araneus pseudoventricosus dragline silk protein gene can be successfully expressed for production of the araneus ventricosus dragline silk protein, and the whole process is simple and efficient, saves the cost, improves the work efficiency, and has good application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. More specifically, it relates to the artificial construction method and application of the traction silk protein gene of E. grandina. Background technique [0002] Spider silk is an excellent natural protein fiber with good properties such as high strength, high elasticity, corrosion resistance, low temperature resistance, lightness, and environmental friendliness, especially traction silk is one of the toughest natural fibers known so far , so spider silk has a wide range of applications in military, medical, industrial, construction, textile and other fields. [0003] However, spiders are cannibalistic and cannot be raised in high density to obtain a large amount of spider silk, and the silk production of spiders is small, so it cannot meet the needs of production and application. Therefore, people hope to use the method of genetic engineering, that is, to transfer the spider silk ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C12N15/10
CPCC12N15/10C07K14/43518
Inventor 王方海刘婷婷
Owner SUN YAT SEN UNIV
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