WO2014026345A1 - Procédé pour l'extraction d'un gène de longueur totale de misp de araneus ventricosus et expression associée - Google Patents

Procédé pour l'extraction d'un gène de longueur totale de misp de araneus ventricosus et expression associée Download PDF

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Publication number
WO2014026345A1
WO2014026345A1 PCT/CN2012/080204 CN2012080204W WO2014026345A1 WO 2014026345 A1 WO2014026345 A1 WO 2014026345A1 CN 2012080204 W CN2012080204 W CN 2012080204W WO 2014026345 A1 WO2014026345 A1 WO 2014026345A1
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WIPO (PCT)
Prior art keywords
misp
length
full
gene
spider
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PCT/CN2012/080204
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English (en)
Chinese (zh)
Inventor
孟清
陈格飞
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东华大学
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Priority to PCT/CN2012/080204 priority Critical patent/WO2014026345A1/fr
Publication of WO2014026345A1 publication Critical patent/WO2014026345A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders

Definitions

  • the present invention relates to the field of genetic engineering technology, and in particular to a method for extracting a full-length gene of MiSp (minor ampul late spidroin) and a full-length MiSp gene expression.
  • each egg contains 500 to 1000 eggs
  • the host mainly harms rice, wheat, cotton, fruit trees and other crops.
  • the abdomen can secrete six kinds of silk protein fibers and one mucus protein.
  • the minor ampul late si lk is a high-quality protein-polymerized fiber with high strength, high elasticity, radiation resistance and heat stability. And biodegradable characteristics, no super-shrinkage in water environment, stable mechanical properties (see Table 1): Table 1 Comparison of performance parameters of spider webs
  • MiSp full-length gene sequence studies have shown that spider silk protein has a highly repetitive primary structure, and its GC content is high, so the full-length gene coding sequence cannot be obtained by PCR alone. Therefore, the cloning of spider silk protein MiSp has been studied so far. The progress is slow.
  • the cDNA library is constructed by cDNA library and hybridization technology to obtain related sequences in the world, but it is only a partial short cDNA sequence of MiSp (see Table 2). So far, there is no MiSp complete genome sequence. Report; Table 2 Partially shorter cDNA sequences of MiSp
  • the existing expression systems including the host: Escherichia coli, Pichia, plants (tobacco, potatoes and Arabidopsis, etc.), mammalian cells, transgenic mice and goats, transgenic silkworms, etc., will be part of the natural gene Expression and artificial synthesis of repetitive module tandem expression, although the expression system is economical, easy to operate, and has the shortest cycle, but due to the problem of codon preference, the protein yield is low or the expression is terminated prematurely and the large molecular weight protein cannot be expressed, and The obtained recombinant incomplete spider silk protein or the artificial splicing tandem body combination protein is far from the natural spider silk protein. In order to make the spider silk protein and fiber large-scale application, it is urgent to realize a complete spider. Silk protein expression technology.
  • the object of the present invention is to overcome the deficiencies of the prior art and to provide a method for extracting the full-length MiSp gene of the large abalone, and realizing the expression of the full MiSp full-length gene.
  • a method for extracting the full-length MiSp gene of the big belly plant was designed. According to this method, the full-length MiSp gene of the large abdomen was obtained, which is characterized by the nucleotide sequence of the full-length MiSp gene of the big belly plant. SEQ ID NO. 1 is shown.
  • the gene is 10929 bp in length and contains two exons of 1428 bp and 3870 bp in length and an intron of 5628 bp in length.
  • a method for extracting a full-length MiSp gene from a spider characterized in that the method comprises the following steps:
  • He 1J was isolated from high Molecular Weight Genomic DNA (HMW-gDNA) by modified CTAB (cetyltriethylammnonium bromide) method;
  • the abdomen fosmid genomic library produced a total of 3. 9 X 10 5 clones, and transferred one by one to about 1026 384-well cell culture plates, and stored at 8% concentration of glycerol at -80 ° C.
  • the 1026 384-well cell culture plates were divided into approximately 26 batches of transfer screening. Each batch was transferred to 40 384-well cell culture plates, and the LB medium was transferred to 40 sterile 384 wells using an 8 X 100 L pipette. a cell culture plate, 60 ⁇ L per well, the LB medium containing 12. 5 g / mL chloramphenicol; B.
  • Construction of the primary mixing tank a) Mix all fosmid genomic library clones in the first batch of 384-well cell culture plates in the first batch with an 8X10 ⁇ 1 pipette, 5 L of bacteria per well, and add to 5 In mL LB medium, the mixture was labeled as No. 1 (384 library clones), and two tubes were aliquoted at 37 ° C and 180 rpm for 5 h. The remaining 39 384-well cell culture plates were cloned in the same manner. Mix, labeled as No. 2 Mix, No. 3 Mix... No. 40 Mix; b) Arrange 40 parts of the mixture obtained in the above procedure in 5 rows and 8 columns, and use 5X 100 ⁇ L pipette to set 5 rows and 8 columns.
  • FD4-16 is a secondary pool (Secondary pools); D. Build a super pool. Mix the first mixture of the first-stage mixing tank with 10 L and add it to 5 mL of LB medium, which is a super pool at 37 °C. Incubate at 180 rpm for 10 h.
  • the CTAB method is a modified CTAB method comprising the following steps: a) grinding: taking the treated spider chest muscles with 89% (v/v) pre-cooled CTAB extraction buffer [2% (g/) V) CTAB, 1. 4 mol/L NaCl, 100 ⁇ ol/L Tris-HCl (pH 8. 0), 20 mmol/L EDTA (pH 8. 0), 2% (g/V) PVP (polyvinylpyrrole) Anthrone)], 1% (V/V) SDS-LDS Master Mix [10%
  • NT+RP and the cleavage protein intron Inl N-terminal domain coding gene with high splicing activity was obtained, namely NT+RP+InlN; the second part of RP 5' The end is fused to the C-terminal domain of Inl, and the RP 3' end is fused to the N-terminal domain of In2, ie, InlC+RP + In2N ; the third part of RP+CT is fused to the C-terminal domain of In2, ie In2 C + RP+CT; (3) The fusion expression product was purified by Ni-NTA.
  • the purified product After mixing the purified NT+RP+InlN with InlC+RP + In2N, InlN and InlC spontaneously undergo trans-splicing reaction, and then self-cleave from the fusion protein, and then carry the MiSp fragment through peptide bond, Ni -NTA reverse purification (removal of Inl), the purified product continues to mix with the third part of In2 C+RP+CT, In2N and In2C are spatially close to each other, spontaneous trans-splicing reaction, and itself is cut from the MiSp fragment, and The MiSp fragments at both ends were linked by peptide bonds, and the recombinant full-length MiSp full-length protein was obtained by a three-step directional splicing reaction.
  • the present invention displays the full MiSp genome sequence of the first large-bellied spider, including the full-length gene of the spider protein MiSp in the large abdomen, and the MiSp full-length gene sequence of the large abalone is more related to other species.
  • This silk protein gene is predicted to be small, but further provides for the expression of the full-length protein.
  • the full-length coding sequence provides gene support for studying the phylogeny of spider silk protein and obtaining full-length recombinant MiSp protein and then bionic high-performance artificial spider silk; in the expression of the big belly plant spider MiSp gene, the present invention adopts protein intron The splicing and Rosetta2 (DE3) expression strains replace the traditional E.
  • the MiSp recombinant protein is segmentally expressed by the trans-splicing reaction of the cleavage protein intron with high splicing activity, through the protein intron Mediated trans-splicing reaction directional splicing of fusion protein in vitro, the recombinant full-length spider silk MiSp protein was obtained, and the natural full-length spider silk protein MiSp gene in Escherichia coli-derived bacteria was completed for the first time at home and abroad.
  • the recombinant expression in the middle provides a material basis for the biomimetic study of spider silk.
  • FIG. 1 is a schematic flow chart of a method for constructing and screening a fosmid library of a large abdomen in the present invention
  • FIG. 2 is a schematic flow chart of a full-length expression method of a large belly plant MiSp according to the present invention
  • 3 is a full-length protein sequence of the Great Spider Mi Mi obtained in the present invention
  • the technical scheme of the present invention mainly extracts the full-length MiSp gene of the spider, and fully expresses the full-length MiSp gene into the full-length protein.
  • the method used to extract the full-length MiSp gene of the big belly spider including the following steps:
  • step d) repeat step d) until the white protein layer is not visible at the interface between the aqueous phase and the organic phase, and the supernatant is taken;
  • the fosmid genomic library of the abdomen garden spiders produced a total of 3. 9 X 10 5 clones, and transferred one by one to about 1026 384-well cell culture plates, and stored at 8% concentration of glycerol at -80 ° C.
  • the 1026 384-well cell culture plates were divided into approximately 26 batches of transfer screens. Each batch was transferred to 40 384-well cell culture plates, and the LB medium was transferred to 40 sterile 384 cells using an 8 X 100 ⁇ L pipette. a cell culture plate, 60 ⁇ L per well, the LB medium containing 12. 5 g / mL chloramphenicol;
  • B. Construction of the primary mixing tank a) Mix all fosmid genomic library clones in the first batch of 384-well cell culture plates in the first batch with an 8 X 10 ⁇ 1 pipette, 5 L of bacteria per well, and add Into 5 mL of LB medium, labeled as No. 1 (384 library clones), and two tubes were incubated at 37 ° C and 180 rpm for 5 h. The remaining 39 384-well cell culture plates were separately prepared in the same manner. Clone and mix, labeled as No. 2 Mix, No. 3 Mix... No.
  • the expression method mainly uses the trans-protein intron-mediated trans-splicing technique to complete the segmental expression of the large abdomen spider MiSp.
  • the protein intron intein
  • the protein intron can spontaneously undergo a splicing reaction, and two The exons on the side are joined by peptide bonds.
  • the two separate polypeptide fragments can be joined to form a complete protein by trans-splicing of the broken protein intron, as follows:
  • NT+RP and the cleavage protein intron Inl N-terminal domain encoding gene with high splicing activity is expressed, ie NT+RP+InlN; the second part of RP 5 ' The end is fused to the C-terminal domain of Inl, and the RP 3 'end is fused to the N-terminal domain of In2, ie, InlC+RP + In2N ; the third part of RP+CT is fused to the C-terminal domain of In2, ie, In2 C + RP+CT;
  • the fusion expression product was purified by Ni-NTA. After mixing the purified NT+RP+InlN with InlC + RP + In2N, InlN and InlC spontaneously undergo trans-splicing reaction, and then self-cleave from the fusion protein, and carry the MiSp fragment through peptide bond, Ni -NTA reverse purification (removal of Inl), the purified product continues to mix with the third part of In2 C+RP+CT, In2N and In2C are spatially close to each other, spontaneous trans-splicing reaction, and itself is cut from the MiSp fragment, and The MiSp fragments at both ends were linked by peptide bonds, and the recombinant full-length MiSp was obtained by a three-step directional splicing reaction.
  • the MiSp full-length protein sequence of the large abalone can be obtained as shown in Figure 3, containing a 5301 bp MiSp full-length transcript, 1766 encoding amino acids, non-repetitive N-terminal and C-terminal modules, and glycine and C.
  • the repeating module consisting of the amino acid consists of the GX, GGX, GGGX and oligo_A repeats motifs and two identical Spacer sequences.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Insects & Arthropods (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé d'extraction du gène de longueur totale de MiSp de Araneus ventricosus et l'expression associée. La séquence nucléotidique du gène de longueur totale obtenue de MiSp de Araneus Ventricosus ayant une longueur totale de 10929pb est telle que présentée dans SEQ ID NO.1, qui contient deux exons ayant une longueur de 1428pb et 387pb, respectivement, et un intron ayant une longueur de 5628pb. Le procédé d'expression du gène de longueur totale de MiSp est au moyen d'un épissage directionnel par l'intermédiaire d'une réaction de trans-épissage qui clive les introns d'une protéine, et l'expression recombinante de la protéine endogène de soie d'araignée de longueur totale du gène MiSp de Araneus ventricosus dans des bactéries issues de E. coli est réalisée et la protéine de longueur totale de MiSp de Araneus ventricosus est obtenue.
PCT/CN2012/080204 2012-08-16 2012-08-16 Procédé pour l'extraction d'un gène de longueur totale de misp de araneus ventricosus et expression associée WO2014026345A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020916A2 (fr) * 2001-08-29 2003-03-13 University Of Wyoming Acides nucleiques codant des proteines de soie d'araignee, polypeptides de soie d'araignee, anticorps specifiques des polypeptides de soie d'araignee et leurs procedes d'utilisation
CN1552854A (zh) * 2003-05-26 2004-12-08 清华大学 一种编码蛛丝蛋白的基因及其应用
CN101679975A (zh) * 2007-03-16 2010-03-24 巴西农牧研究企业 来自Nephilengys Cruentata蜘蛛、黄带粉趾食鸟蛛和Parawixia Bistriata蜘蛛的网的蛋白

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003020916A2 (fr) * 2001-08-29 2003-03-13 University Of Wyoming Acides nucleiques codant des proteines de soie d'araignee, polypeptides de soie d'araignee, anticorps specifiques des polypeptides de soie d'araignee et leurs procedes d'utilisation
CN1552854A (zh) * 2003-05-26 2004-12-08 清华大学 一种编码蛛丝蛋白的基因及其应用
CN101679975A (zh) * 2007-03-16 2010-03-24 巴西农牧研究企业 来自Nephilengys Cruentata蜘蛛、黄带粉趾食鸟蛛和Parawixia Bistriata蜘蛛的网的蛋白

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN GEFEI: "Cloning and characterization of araneus ventricosus Masp gene based on Fosmid genomic library and STS/3D-PCR", CHINA EXCELLENCE MASTER THESIS FULL TEXT DATABASE, 10 June 2010 (2010-06-10) *
JOHN GATESY ET AL.: "Extreme Diversity, Conservation, and Convergence of Spider Silk Fibroin Sequences", SCIENCE, vol. 291, no. 5513, 30 March 2001 (2001-03-30), pages 2603 - 2605, XP001191057, DOI: doi:10.1126/science.1057561 *

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