WO2014026345A1

WO2014026345A1 - Method for extracting full-length gene of misp of araneus ventricosus and expressing same

Info

Publication number: WO2014026345A1
Application number: PCT/CN2012/080204
Authority: WO
Inventors: 孟清; 陈格飞
Original assignee: 东华大学
Priority date: 2012-08-16
Filing date: 2012-08-16
Publication date: 2014-02-20

Abstract

Disclosed is a method for extracting the full-length gene of MiSp of Araneus ventricosus and expressing same. The nucleotide sequence of the obtained full-length gene of MiSp of Araneus ventricosus with a full length of 10929bp is as shown by SEQ ID NO.1, which contains two exons with a length of 1428bp and 3870bp, respectively, and an intron with a length of 5628bp. The expression method of the full-length gene of MiSp is by means of directional splicing via a trans-splicing reaction which cleaves the introns of a protein, and the recombinant expression of the native full-length spider silk protein MiSp gene of Araneus ventricosus in bacteria derived from E. coli is completed and the full-length protein of MiSp of Araneus ventricosus is obtained.

Description

Method for extracting full-length gene and full-length gene expression of MiSp

[Technical Field] 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.

[Background Art] Large abdomen spider (ΑΓ3 deleted s ventricosus) belongs to the genus Araneae, arachnid, mainly distributed in Hubei, Hunan, Sichuan, Jiangsu, Zhejiang, Anhui, Guizhou, Yunnan, Henan, Hebei Qinghai, Xinjiang. Etc., the species is large in size and grayish brown. It is used to form a large wheel-shaped vertical circular net between eaves, gardens and trees. The center of the night net is concealed in the gaps or leaves in the net during the day. The egg pouch is produced. Walls or bark cracks, etc., 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

Material Strength (N m-2) Elongation (%) Energy to break (J kg-1)

Dragline silk 4x l0 ⁹ 35 4>< 10 ⁵

Minor ampullate silk lx lO ⁹ 5 3>< 10 ⁴

Kevlar 4x l0 ⁹ 5 3>< 10 ⁴

Tendon lx lO ⁶ 5 5>< 10 ³

Some data from Gosline, JM; Dennv, MW; DeMont, ME Nature 1984, 309, 551. Therefore, this silk protein fiber has a huge field in military, aerospace and biomedical engineering. The potential application value, but because the spider has intra-species residual nature, the natural silk production is much lower than that of the silkworm. The domestication of the spider silk is inefficient, so the genetic engineering technology is used to synthesize the spider silk protein and then spun into a spider silk to become a bionic spider. The only way to develop silk. The first step in the synthesis of temporary capture silk using genetic engineering techniques is to obtain the spider protein of the abdomen

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

Species cDNA/DNA Length (bp) Accession NO. Year

Nephila clavipes cDNA 3060 AF027735 1998

Nephila clavipes cDNA 855 AF027736 1998

Nephila clavipes cDNA 472 AF027737 1998

Nephila ntipodiana cDNA 1189 DQ338462 2006

Latrodectus esperus cDNA 1585 EU394445 2008

Latrodectus esperus cDNA 1043 HM752571 2010

Latrodectus esperus cDNA 973 HM752570 2010

Argiope argentata cDNA 540 JQ713003 2012

Argiope argentata cDNA 1195 JQ713004 2012

Metepeira randiosa cDNA 1566 HM752575 2010

Metepeira andiosa cDNA 1160 HM752569 2010

Uloborus diversus cDNA 1550 HM752574 2010

Uloborus diversus cDNA 2646 DQ399332 2006

Deinopis spinosa cDNA 1604 DQ399324 2006

Parawixia bistriata cDNA 1709 GQ275358 2011

Parawixia bistriata cDNA 1754 GQ275359 2012

Parawixia bistriata cDNA 1434 GQ275360 2012 The recombination of the spider protein of the Abdominal Garden is based on the obtained full-length gene sequence of the spider silk protein MiSp of the large abdomen, but due to the large and severe gene encoding the spider silk protein and the high GC content of the coding gene, The gene is genetically unstable in the expression host, and it is easy to form a stable mRNA secondary structure, etc., resulting in a base. Due to many problems such as early termination of transcription, deletion, gene rearrangement, and early termination of translation, the existing expression system is unable to complete the full-length expression of this ultra-high molecular weight protein. 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.

SUMMARY OF THE INVENTION 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. In order to achieve the above objectives, 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:

(1) Degenerate PCR to obtain MiSp C-terminal gene sequence A. Analyze the C-terminal amino acid sequence of the traced silk protein MaSp and the temporary capture silk protein MiSp in the well-known spider web, and obtain the conserved amino acid sequences, namely SRISSA and NIGQVD;

B. Designing the upstream and downstream primers of degenerate PCR based on the two conserved amino acid sequences of SRISSA and NIGQVD;

C. Using the high molecular weight genomic DNA of spider web of Daweiyuan as a template, the gene fragment sequence of the MiSp C-terminal length of about 200 bp was obtained by degenerate PCR and sequencing analysis.

D. Based on the sequence of the gene fragment of the MiSp C-terminal length of about 200 bp, design specific primers (GMiSp-CF: 5' - TTACTCAGGTGTCCTTGG-3, GMiSp-CR: 5' - ATTGGCTTACTGCATTCT - 3') for library screening;

(2) Separating high molecular weight DNA to construct fosmid genomic library

A. He 1J was isolated from high Molecular Weight Genomic DNA (HMW-gDNA) by modified CTAB (cetyltriethylammnonium bromide) method;

B. Constructing a fosmid genomic library of the large abalone plant using a library construction kit;

(3) Screening fosmid genomic library

A. 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 μL 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 μ1 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 μL pipette to set 5 rows and 8 columns. Five wells in each column of the array were mixed, and the mixed 8 groups of bacteria were extracted from each well by 5 41^ into 511^1^ medium, numbered FD1-1, FD1-2-FD1-8, respectively. Incubate at 37 °C, 180 rpm for 10 h, and mix each row of 5 rows and 8 columns in the same way, and number them as FD2-1, FD2- 2-FD2- 5, FD1- 1, FD1- 2-FD1- 8 and FD2- 1, FD2- 2··· FD2-5 obtain the primary pools;

C. Construct a secondary mixing tank a) Pipette 5 L of each of the first batch of 40 384-well cell culture plates in the first batch of 8 10 10 μ 1 pipette into 5 mL LB medium, label FD3-1, incubated at 37 °C, 180 rpm for 10 h, mix the 2nd to 24th columns in the same way, labeled FD3-2, FD3_3 "03-24, a total of 24 mixing tanks; b) press the same Methods The AP lines of 40 384-well cell culture plates were mixed, numbered FD4-1, FD4-2--FD4-16, a total of 16 mixing cells, FD3_1, FD3-2-FD3-24 and FD4_1, FD4. -2—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.

(4) Sequencing of positive clones The positive clones containing the full-length gene were subjected to Shotgun sequencing, and the sequencing coverage was set to about 7, and finally verified by PCR, restriction enzyme digestion and sequencing, and the MiSp full-length gene sequence of the spider was obtained. 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 匪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%

(g/V) SDS, 10% (g/V) LDS] and 1% (V/V) β-mercaptoethanol in a 1.5 mL centrifuge tube, fully ground and crushed; b) Water bath: 65 °C water bath approx. 2 h, from time to time, gently upside down until the mixture is digested very clearly; c) Extraction: Add an equal volume of phenol-chloroform-isoamyl alcohol (25: 24: 1), gently mix by inversion, centrifuge at high speed on a table Centrifuge at 12000 rpm for 5 min on the machine, take the supernatant; d) Extract with chloroform: Add equal volume of chloroform to the supernatant, gently mix by inversion, centrifuge at 12000 rpm for 5 min on a benchtop high-speed centrifuge. Supernatant e) Repeat step d) until the white protein layer is not visible at the interface between the aqueous phase and the organic phase, and take the supernatant; f) Precipitate the DNA: Add 1/10 volume of 5 mol/L NaCl to the supernatant. And 2.5 times absolute ethanol (pre-cooling), gently mix, place the pellet at -20 °C for 30 min, centrifuge at 13,000 rpm for 15 min on a benchtop high-speed centrifuge, discard the supernatant; g) wash the DNA: The precipitated centrifuge tube was washed with 1 mL of 70% ethanol, centrifuged at a speed of 13000 111 on a benchtop high-speed centrifuge, and the supernatant was discarded once. h) Preservation: Dry the precipitate at room temperature for 5 min, add The appropriate amount of TE buffer [10 ol / L of Tris_HCl (pH 8.0) and 1 mmol / L of EDTA (pH 8.0)] dissolved DNA, stored at - 20 °. Or spare at 4 °C. A method for expressing the MiSp full-length gene of the large abdomen, characterized in that the method is to perform segmental expression of the spider, Mi Sp, by using a protein intron-mediated trans-splicing technique, which is mediated by protein introns. The trans-splicing technique is used for directional splicing in vitro to achieve complete expression of the full-length MiSp of the large abdomen. The specific steps are as follows:

(1) Divided the full-length MiSp gene into three parts of the Rosetta2 (DE3) expression strain: NT+RP RP and RP+CT;

(2) Using DNA recombination technology, the expression of 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. 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. Compared with the prior art, 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. coli as the host cell expression technology, and 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.

BRIEF DESCRIPTION OF THE DRAWINGS 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; [Detailed Description of the Invention] The method of the present invention will be further described with reference to the accompanying drawings, and the method steps thereof can be realized by those skilled in the art. 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:

(1) Degenerate PCR to obtain MiSp C-terminal gene sequence

A. Analyze the C-terminal amino acid sequence of the traced silk protein MaSp and the temporary capture silk protein MiSp in the well-known spider web, and obtain the conserved amino acid sequences, namely SRISSA and NIGQVD;

D. Based on the gene fragment sequence of the MiSp C-terminal length of about 200 bp, design specific primers (GMiSp-CF: 5' - TTACTCAGGTGTCCTTGG-3, GMiSp-CR: 5' - ATTGGCTTACTGCATTCT - 3') for library screening;

(2) Separating high molecular weight DNA to construct a fosmid genomic library, as shown in Figure 1: A. Hejl uses the modified CTAB (cetyltriethylammnonium bromide) method to isolate High Molecular Weight Genomic DNA (HMW-gDNA); the steps of the modified CTAB method: a) Grinding. The treated spider chest muscles were loaded with 89% (v/v) pre-cooled CTAB extraction buffer [2% (g/V) CTAB, 1.4 mol/L NaCl, 100 mmol/L Tris-HCl (pH 8. 0), 20 mmol/L EDTA (pH 8.0), 2% (g/V) PVP (polyvinylpyrrolidone)], 1% (V/V) SDS-LDS master mix [10% (g/V) SDS, 10% (g/V) LDS] and 1% (V/V) β-mercaptoethanol in a 1.5 mL centrifuge tube, fully ground and crushed;

b) Water bath. The water bath at 65 °C is about 2 hours, during which time it is gently inverted up and down until the mixture is digested very clearly; c) extracted. Add an equal volume of phenol-chloroform-isoamyl alcohol (25: 24: 1), gently mix by inversion, centrifuge at a speed of 12000 rpm for 5 min on a bench-top high speed centrifuge, and take the supernatant;

d) Extraction by chloroform. Add equal volume of chloroform to the supernatant, gently mix by inversion, centrifuge at 12000 rpm for 5 min on a benchtop high-speed centrifuge, and take the supernatant;

e) 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;

f) Precipitating DNA. Add 1/10 volume of 5 mol/L NaCl and 2.5 times absolute ethanol (pre-cool) to the supernatant, mix gently, place the pellet at -20 °C for 30 min, and centrifuge at 13000 rpm for 15 min on a benchtop high-speed centrifuge. Discard the supernatant;

g) Wash DNA. The DNA was washed by adding 1 mL of 70% ethanol to the centrifuge tube with the precipitate, and centrifuged at 13,000 rpm for 5 min in a benchtop high-speed centrifuge, and the supernatant was discarded and repeated once;

h) Save. Dry the precipitate at room temperature for 5~10 min, add appropriate amount of TE buffer [10 mmol/L Tris-HCl

(pH 8.0) and 1 mmol/L EDTA (pH 8.0)] Dissolve the DNA and store at -20 or 4 °C until use. B. Constructing a fosmid genomic library of the large abalone plant using a library construction kit;

(3) Screening the fosmid genomic library, as shown in Figure 1:

A. 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 μL pipette. a cell culture plate, 60 μL 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 μ 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. 40 Mix; b) Place 40 parts of the mixture obtained in the above procedure in 5 rows x 8 columns, using 8 x 100 μL pipette 5 rows 五个 Five wells in each column of the 8 column arrangement were mixed, and the mixed 8 groups of bacteria were extracted 5 times into each well and added to 5 mL of LB medium, numbered FD1_1, FD1-2-FD1-8, respectively. Incubate at 37 °C, 180 rpm for 10 h, mix each row of 5 rows and 8 columns in the same way, and number them as FD2-1, FD2-2--FD2-5. FD1- 1, FD1 -2'"FD1_8 and FD2_1, FD2- 2··· FD2-5 obtain the primary pools (Primary pools);

C. Building a secondary mixing pool a) Pipette 5 μL of each of the first batch of 40 384-well cell culture plates in column 1 with an 8 X 10 μ 1 pipette and add to 5 mL of LB medium, label FD3-1, Incubate at 37 °C, 180 rpm for 10 h, mix the 2nd to 24th columns in the same way, labeled FD3- 2, FD3- 3-FD3- 24, a total of 24 mixing tanks; b) 40 in the same way The AP line of the 384-well cell culture plate was mixed and numbered as FD4- 1 , FD4-2 · -FD4-16, a total of 16 mixing cells, FD3- 1, FD3- 2 · -FD3-24 and FD4- 1 FD4- 2 · · · FD4-16 is a secondary pool (Secondary pools);

D. Build the super pool. Mix the first mixture of the first-stage mixing tank with 10 μL and add it to 5 mL of LB medium, which is the super pool at 37 °C. Incubate at 180 rpm for 10 h.

(4) Sequencing of positive clones The positive clones containing the full-length gene were subjected to Shotgun sequencing, and the sequencing coverage was set to about 7. Finally, the full-length MiSp gene sequence of the large abalone was obtained by PCR, restriction enzyme digestion and sequencing. The nucleotide sequence of the MiSp full-length gene obtained by the above method is shown in SEQ ID NO. 1, which is 10929 bp in length and contains two exons of lengths of 1428 bp and 3870 bp, respectively. An intron of 5628 bp in length. In order to obtain the MiSp full-length protein of the large abalone, it is necessary to express the full-length MiSp gene of the large abalone, and the expression method mainly uses the trans-protein intron-mediated trans-splicing technique to complete the segmental expression of the large abdomen spider MiSp. In vitro directional splicing using protein intron-mediated trans-splicing technology to achieve full expression of the full-length protein of the MiSp spider, the protein intron (intein) is present in the precursor egg An insertion sequence in a precursor protein, the amino acid sequence on both sides of which is called an extein. In the process of forming a mature protein, 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:

(1) Divided the full-length MiSp gene into three parts of Rosetta2 (DE3): NT+RP, RP and RP+CT;

(2) Using DNA recombination technology, the expression of 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;

(3) 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. According to the above steps, 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.

Claims

A full length MiSp full-length gene, characterized in that the nucleotide sequence of the MiSp full-length gene of the large abalone is as shown in SEQ ID NO.

The full-length MiSp gene of the large-bellied spider, according to claim 1, characterized in that the gene is 10929 bp in length and comprises two exons of length 1428 bp and 3870 bp, respectively, and a length of 5628. Intron of bp.

A method for extracting a MiSp full-length gene of the large abdomen spider according to claim 1 or 2, characterized in that the method comprises the following steps:

(1) Degenerate PCR to obtain MiSp C-terminal gene sequence

B. Designing upstream and downstream primers for degenerate PCR based on the two conserved amino acid sequences of SRISSA and NIGQVD;

(2) Separating high molecular weight DNA to construct fosmid genomic library A. Using the CTAB (cetyltriethylammnonium bromide) method to isolate high Molecular Weight Genomic DNA (HMW-gDNA);

B. Using the library construction kit to construct the fosmid genomic library; (3) screening the fosmid genomic library

A. The large abdomen spider fosmid genomic library produced a total of 3. 9 X 10 ⁵ clones, and transferred one by one

1026 384-well cell culture plates were stored at -80 ° C with 8% concentration of glycerol. The 1026 384-well cell culture plates were divided into about 26 batches of transfer screening, and 40 384-well cell culture plates were transferred per batch. 5 μg/毫升氯霉素; The LB medium is transferred to a 40 sterilized 384-well cell culture plate, 40 L per well, and the LB medium contains 12. 5 μg/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 μ 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. 40 Mix; b) Arrange 40 parts of the mixture obtained in the above procedure in 5 rows and 8 columns, using 8 x 100 μL pipette Five wells in each of the 5 rows and 8 columns are mixed, and the mixed 8 groups of bacteria are extracted 5 4 per well, and added to the 5 1 ^ 1 ^ medium, numbered FD1-1, FD1-2, respectively. — FD1—8, culture at 37 °C, 180 rpm for 10 h, in the same way, arrange 5 rows of X 8 columns. Each row is mixed and numbered FD2-1, FD2-2-FD2-5, FD1- 1, FD1- 2··· FD1-8 and FD2-1, FD2-2'"FD2_5 to obtain the primary mixing Pool (Primary pools);

C. Construction of two mixing tank a) with ₈ X 10 μ ι pipetted broth 384 40-well cell culture plate in a first batch of each of the first column of 5 L, were added to 5 mL LB medium, Label FD3-1, cultured at 37 °C, 180 rpm for 10 h, mixing the 2nd to 24th columns in the same way, labeled FD3_2, FD3-3--FD3-24, a total of 24 mixing cells; b) The AP lines of 40 384-well cell culture plates were mixed in the same manner, numbered as FD4- 1 , FD4-2 · · · FD4- 16, a total of 16 mixing cells, FD3- 1 , FD3-2 · -FD3 -24 and FD4- 1, FD4-2'" FD4-16 is a secondary pool (Secondary pools);

(4) Sequencing of positive clones The positive clones containing the full-length gene were subjected to Shotgun sequencing, and the sequencing coverage was set to about 7, and finally verified by PCR, restriction enzyme digestion and sequencing, and the MiSp full-length gene sequence of the spider was obtained.

The method for extracting the MiSp full-length gene of the large-bellied spider, according to claim 3, wherein the CTAB method is a modified CTAB method, and the method comprises the following steps: a) Grinding: Take the treated spider chest muscles with 89% (V/V) pre-cooled CTAB extraction buffer [2% (g/V) CTAB, 1. 4 mol/L NaCl, 100 mmol/L Tris-HCl (pH 8. 0), 20 mmol/L EDTA (pH 8. 0), 2% (g/V) PVP (polyvinylpyrrolidone)], 1% (V/V) SDS-LDS Master Mix [10%

(g/V) SDS, 10% (g/V) LDS] and 1% (V/V) β-mercaptoethanol in a 1.5 mL centrifuge tube, fully ground and crushed; b) Water bath: 65 °C water bath approx. 2 h, from time to time, gently upside down until the mixture is digested very clearly; c) Extraction: Add an equal volume of phenol-chloroform-isoamyl alcohol (25: 24: 1), gently mix by inversion, centrifuge at high speed on a table Centrifuge at 12000 rpm for 5 min on the machine, take the supernatant; d) Extract with chloroform: Add equal volume of chloroform to the supernatant, gently mix by inversion, centrifuge at 12000 rpm for 5 min on a benchtop high-speed centrifuge. Supernatant; e) Repeat step d) until the white protein layer is not visible at the interface between the aqueous phase and the organic phase, and take the supernatant; f) Precipitate DNA: Add 1/10 volume to the supernatant. Mol/L NaCl and 2.5 times absolute ethanol (pre-cooled), gently mix, precipitate the DNA at -20 °C for 30 min, centrifuge at 13,000 rpm for 15 min on a benchtop high-speed centrifuge, discard the supernatant; g) Wash DNA: Wash the DNA by adding 1 mL of 70% ethanol to the centrifuge tube with the precipitate, and centrifuge at a speed of 13000 rpm for 5 min on a benchtop high-speed centrifuge. Clear solution, repeat 1 time; h) Storage: Dry the precipitate at room temperature for 5~10 min, add appropriate amount of TE buffer [10 mmol/L Tris_HCl (pH 8.0) and 1 mmol/L EDTA (pH 8.0)] DNA, stored at - 20 °. Or spare at 4 °C.

A method for expressing a MiSp full-length gene of the large abdomen spider according to claim 1 or 2, wherein the method comprises the step of expressing the large abdomen by means of a broken protein intron-mediated trans-splicing technique. Spider MiSp, using protein intron-mediated trans-splicing technology to direct splicing in vitro, to achieve full expression of the full-length MiSp of the spider, the specific steps are as follows:

(1) Divided the full-length MiSp gene into three parts of the Rosetta2 (DE3) expression strain: NT+RP, RP and RP+CT;

(2) Using DNA recombination technology, the expression of 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 3' end of RP is expressed in fusion with the N-terminal domain of In2, ie, InlC + RP + In2N _{; the} third part of RP+CT is fused with the C-terminal domain of In2, ie In2 C + RP+CT;

(3) 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 cut 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, itself 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.