CN106754946A - A kind of artificial constructed Araneus ventricosus dragline silk protein and its construction method - Google Patents
A kind of artificial constructed Araneus ventricosus dragline silk protein and its construction method Download PDFInfo
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Abstract
The invention discloses a kind of artificial constructed Araneus ventricosus dragline silk protein and its construction method.The sequence of the Araneus ventricosus dragline silk protein is as shown in SEQ ID NO.6.The present invention is first according to Araneus ventricosus traction fiber protein part cDNA sequence, choose most representative core repeat sequence 108bp, carry out artificial synthesized, and add restriction enzyme site at the core repeat sequence two ends, then it is connected and converts to Escherichia coli with the EcoR V/BAP carriers of pSIMPLE 19 and obtains monomer recombinant plasmid, then gene monomer poly is turned to by 16 times of concatermers using the end to end method of isocaudarner, intend Araneus ventricosus dragline silk protein so as to have successfully been obtained 1836bp, can successful expression production Araneus ventricosus traction silk-fibroin, whole flow process simple and effective, it is not only cost-effective, and improve operating efficiency, application prospect is good.
Description
Technical field
The invention belongs to gene engineering technology field.More particularly, to the artificial of Araneus ventricosus dragline silk protein
Construction method and its application.
Background technology
Spider silk is a kind of excellent natural protein fiber, with high intensity, high resiliency, it is corrosion-resistant, low temperature resistant, slim and graceful,
The good performance of environmentally friendly grade, especially traction fiber are one of natural fibers the most tough and tensile for being currently known, therefore spider
The spider's thread has a wide range of applications in fields such as military affairs, medical science, industry, building, weavings.
But spider cannibalism, it is impossible to high-density rearing is to obtain substantial amounts of spider silk, and the product silk amount of spider is few, because
And the need for production application can not being met, then it is desirable to using genetic engineering method, will spider silk gene be transferred to not
Spidroins are expressed in same organism, because spider silk protein gene is huge repetitive sequence, full-length gene sequence is processed
Row are extremely difficult, therefore artificial constructed macromolecule spider silk gene is one of important step of this kind of work.It is artificial in the world at present
It is network bride category spider (Nephlia clavipe) traction fiber protein sequence to build most spider silk genes, is existed respectively
Expression is attempted in the biology such as Escherichia coli, yeast, tobacco, potato, milk cow and silkworm.
Araneus ventricosus are widely distributed in China, are common spider kind, and larger and silk performance of knotting is protruded very much.But at present
Correlative study and the report of artificial constructed Araneus ventricosus macromolecule dragline silk protein are not related to also.
The content of the invention
The technical problem to be solved in the present invention is the defect and deficiency for overcoming above-mentioned prior art, there is provided a kind of artificial constructed
Araneus ventricosus macromolecule dragline silk protein, be from now on using gene engineering method produce Araneus ventricosus macromolecule traction fiber
Albumen provides target gene, serves produce reality needs.
It is an object of the invention to provide a kind of method of artificial constructed Araneus ventricosus dragline silk protein.
Another object of the present invention is to provide a kind of artificial constructed Araneus ventricosus dragline silk protein.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of sequence monomer of Araneus ventricosus dragline silk protein, its sequence is as shown in SEQ ID NO.2.
One kind intends Araneus ventricosus dragline silk protein, and its sequence is as shown in SEQ ID NO.5.
A kind of artificial constructed Araneus ventricosus traction silk-fibroin, the sequential coding translation as shown in SEQ ID NO.5 is obtained.
In addition, SEQ ID NO.1, sequence shown in SEQ ID NO.2 or SEQ ID NO.5 are led in artificial constructed Araneus ventricosus
Draw the application in terms of Silk gene, also within protection scope of the present invention.
A kind of method of artificial constructed Araneus ventricosus dragline silk protein, comprises the following steps：
（1）Sequence shown in SEQ ID NO.2 is divided into 2 ends 18 72bp fragments of base complementrity, and synthesis obtains two
Oligonucleotide chain, by complementation connection filling-in, obtains double-stranded sequence；The double-stranded sequence is connected to pSIMPLE-19 EcoR
V/BAP expression vectors, obtain pSIMPLE-19 EcoR V/BAP monomer recombinant plasmids；
（2）Recombinant plasmid is respectively through Nhe I/Hind III double digestions and Spe I/Hind III double digestions, two digestions
Product is connected, and obtains 2 times of concatermers of Araneus ventricosus dragline silk protein core repeat sequence；The step is repeated, big abdomen is obtained
16 times of concatermers of epeira dragline silk protein core repeat sequence, as artificial constructed Araneus ventricosus draw the volume of silk-fibroin
Code sequence, as shown in SEQ ID NO.5.
Particularly preferably, the method for above-mentioned artificial constructed Araneus ventricosus dragline silk protein, comprises the following steps：
S1. the sequence monomer of Araneus ventricosus dragline silk protein is prepared
S11. according to Araneus ventricosus macromolecule traction fiber protein part cDNA sequence, core repeat sequence, such as SEQ ID are determined
Shown in NO.1（108bp）, constituted as the basi gene of monomer；
S12. 5 of core repeat sequence shown in SEQ ID NO.1’End is plus Nhe I restriction enzyme sites GCTAGC, 3’End adds
Spe I restriction enzyme site ACTAGT and Hind III digestion site AAGCTT, obtain the monomer of Araneus ventricosus dragline silk protein
Sequence, as shown in SEQ ID NO.2（126bp）；
S13. gene monomer sequence shown in above-mentioned SEQ ID NO.2 is divided into 2 fragments of 72bp, the end of two fragments has
18 base complementrities；Sequent synthesis according to two fragments obtain two oligonucleotide chains；
S14. two synthetic oligonucleotide chains are carried out into complementary connection, and filling-in is carried out by PCR reactions, obtain 2 ends
The double-stranded sequence of the Araneus ventricosus dragline silk protein monomer with restriction enzyme site；
S2. pSIMPLE-19 EcoR V/BAP monomer recombinant plasmids are built
S21. the double-stranded sequence of above-mentioned Araneus ventricosus dragline silk protein monomer is carried out into phosphorylation；
S22. the product of phosphorylation is attached with pSIMPLE-19 EcoR V/BAP expression vectors again；
S23. by connection product transformed competence colibacillus cell, by the screening and identification of transformant, pSIMPLE-19 EcoR are obtained
V/BAP monomer recombinant plasmids；
S3. Araneus ventricosus many times of concatermers of dragline silk protein core repeat sequence are built
S31. culture conversion has the bacterium solution of pSIMPLE-19 EcoR V/BAP monomer recombinant plasmids, extracts pSIMPLE-19
EcoR V/BAP monomer recombinant plasmids；
S32. pSIMPLE-19 EcoR V/BAP monomers recombinant plasmid is used into restriction enzyme Nhe I/Hind respectively
III double digestions, with restriction enzyme Spe I/Hind III double digestions, be separately recovered and obtain genes of interest and linear plasmid
Carrier；
S33. the genes of interest fragment and linear plasmid carrier of recovery are attached with T4 DNA ligases；Connection product turns
Change competent cell；
S34. positive recombinant is screened with bacterium solution PCR methods, so as to obtain Araneus ventricosus dragline silk protein core repeat sequence
2 times of concatermers；
S35. Araneus ventricosus dragline silk protein 2 times of concatermer plasmids of core repeat sequence are extracted, above-mentioned S32-S34 is repeated
Method, obtain Araneus ventricosus dragline silk protein 16 times of concatermers of core repeat sequence, as artificial constructed great Fu gardens
Spider draws the coded sequence of silk-fibroin, as shown in SEQ ID NO.5.
Wherein it is preferred to, the reaction volume of PCR reactions is 25 μ L described in step S14, and reaction system is included：5×
PrimeSTAR buffer solutions, 200 μM of dNTPs, 0.75 U PrimeSTAR HS DNA polymerases.
Preferably, PCR reaction conditions described in step S14（That is filling-in condition）For：94 DEG C of predegeneration 2min；94 DEG C of denaturation
1min, 49 DEG C of annealing 30s, 72 DEG C of extension 20s, circulates 4 times；Last 72 DEG C of extensions 10min.
Preferably, the method for phosphorylation is described in step S21：Take the double-strand sequence of Araneus ventricosus dragline silk protein monomer
Arrange into row agarose gel electrophoresis, after carrying out rubber tapping recovery to the purpose band of 126bp, carry out phosphatizing treatment.
Preferably, the reaction system of phosphorylation is：ddH2O 10.25μl、10×T4 DNA Polynucleotide
The μ l of Kinase Buffer 2.5, the μ l of filling-in product 12 of above-mentioned recovery, ATP 25nmol (0.25 μ l), T4
Polynucleotide Kinase 10units。
Preferably, the reaction condition of phosphorylation is：Reacted 30 minutes at 37 DEG C.
Preferably, the reaction system of connection is described in step S22：The μ l of pSIMPLE-19 EcoR V/BAP expression vectors 1,
Insert DNA (above-mentioned Phosphorylated products) 4 μ l, Solution I (DNA Ligation Kit Ver.2.1) 5 μ l.It is excellent
Selection of land, the reaction condition of connection is：2h is reacted at 16 DEG C.
Preferably, the specific method of step S23 is：Connection product is transformed into bacillus coli DH 5 alpha competent cell,
Containing Amp（It is preferred that 100 μ g/mL）Selective flat board on be coated with bacterium solution, cultivate 15~18 h, picking white colony, inoculation
To containing Amp（It is preferred that 100 μ g/mL）B fluid nutrient mediums in, more than 37 DEG C of shaking table shaken cultivation 6h；Take bacterium solution and enter performing PCR
Identification.
Preferably, the reaction volume that the PCR is identified is 25 μ L, Premix Taq enzymes, universal primer M13F and M13R
（Sequence is respectively as shown in SEQ ID NO.3 and SEQ ID NO.4）；
Preferably, the reaction condition that the PCR is identified is：94 DEG C of predegeneration 1min；94 DEG C of denaturation 25s, 49 DEG C of anneal 25s, 72
DEG C extend 30s, circulate 32 times；Last 72 DEG C of extensions 10min.
Preferably, the reaction system of double digestion is described in step S32：The μ g of plasmid 2, the μ L of reaction buffer 5, each 1 μ of restriction endonuclease
L、ddH2O is mended to 50 μ L.
Preferably, the reaction condition of double digestion is described in step S32：Reaction system gently mix after brief centrifugation, 37 DEG C
Insulation 2h.
Preferably, competent cell described in step S23 or S33 is bacillus coli DH 5 alpha competent cell.
The present invention chooses most representative core and repeats first according to Araneus ventricosus traction fiber protein part cDNA sequence
Sequence 108bp, carry out it is artificial synthesized, and its 5’End adds Nhe I restriction enzyme sites, 3’End adds Spe I and Hind III enzymes
Enzyme site, is then connected and converts to Escherichia coli and obtain monomer recombinant plasmid with pSIMPLE-19 EcoR V/BAP carriers,
Then gene monomer poly is turned to by 16 times of concatermers using the end to end method of isocaudarner, intends big so as to have successfully been obtained 1836bp
The coded sequence of abdomen epeira dragline silk protein.
The invention has the advantages that：
The present invention chooses most representative core repeat sequence first according to Araneus ventricosus traction fiber protein part cDNA sequence
108bp, carry out it is artificial synthesized, and the end of sequence 2 add restriction enzyme site, then with pSIMPLE-19 EcoR V/BAP carriers connect
Connect and convert to Escherichia coli and obtain monomer recombinant plasmid, then using the end to end method of isocaudarner by gene monomer multimerization
It is 16 times of concatermers, so as to have successfully been obtained the coded sequence that 1836bp intends Araneus ventricosus dragline silk protein, whole flow process
Simple and effective, it is not only cost-effective, and improve operating efficiency.
Brief description of the drawings
Fig. 1 is the flow chart of the core repeat sequence monomer that the present invention prepares Araneus ventricosus dragline silk protein.
Fig. 2 is the sequence monomer of the Araneus ventricosus dragline silk protein of preparation in the present invention；Wherein, overstriking sequence in square frame
It is classified as Nhe I restriction enzyme sites；Overstriking sequences in italics is Spe I restriction enzyme sites in square frame, and sequences in italics is the restriction enzyme sites of Hind III.
Fig. 3 is clone and the dliploidization schematic diagram of Araneus ventricosus dragline silk protein monomer of the present invention.
Fig. 4 is pSIMPLE-19 EcoR V/BAP monoploid recombinant plasmid bacterium solution PCR qualification figures.
Fig. 5 is that figure is reclaimed in the digestion of pSIMPLE-19 EcoR V/BAP monoploid recombinant plasmids；2nd swimming lane is recombinated for monomer
Plasmid restriction enzyme Nhe I/Hind III double digestions, it is seen that the genes of interest fragment band of 124b or so；3rd
Swimming lane is monomer recombinant plasmid restriction enzyme Spe I/Hind III double digestions, it is seen that the linear load of 2816bp or so
Body band.
Fig. 6 is 3 PCR qualification figures of two times of concatermer recombinant plasmid clones.
Fig. 7 is 4 times of PCR qualification figures of concatermer recombinant plasmid clone.
Fig. 8 is 8 times of PCR qualification figures of concatermer recombinant plasmid clone.
Fig. 9 is 16 times of PCR qualification figures of concatermer recombinant plasmid clone.
Figure 10 is artificial constructed plan Araneus ventricosus dragline silk protein.
Specific embodiment
The present invention, but embodiment are further illustrated below in conjunction with Figure of description and specific embodiment not to the present invention
Limit in any form.Unless stated otherwise, reagent, the method and apparatus that the present invention is used are for the art is routinely tried
Agent, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention and material are purchased in market.
Embodiment 1 prepares the sequence monomer of Araneus ventricosus dragline silk protein
Flow chart is as shown in Figure 1.
1st, according to Araneus ventricosus traction fiber protein part cDNA sequence (JN857964.2) delivered, determine that core is repeated
Sequence 108bp（As shown in SEQ ID NO.1）Constituted as the basi gene of monomer.
Then the 5 of above-mentioned core repeat sequence’End adds Nhe I restriction enzyme site GCTAGC, in Spe I and Hind
III digestion site ACTAGTAAGCTT, so the sequence total length of composition are 126bp（As shown in SEQ ID NO.2）, as greatly
The sequence monomer of abdomen epeira dragline silk protein.
From the two ends of gene monomer, artificial synthesized two oligonucleotides are single-stranded（72bp, sequence is as shown in SEQ ID NO.3）,
Two there are 18 base complementrities the single-stranded end of oligonucleotides.
2nd, by two synthetic oligonucleotide chains（Synthesized by Takara companies）Respectively taking 100ng carries out complementary connection,
And filling-in is carried out by PCR reactions, reaction volume is 25 μ L, 5 × PrimeSTAR buffer solutions, 200 μM of dNTPs, 0.75 U
PrimeSTAR HS DNA polymerases, filling-in condition：94 DEG C of predegeneration 2min；94 DEG C of denaturation 1min, 49 DEG C of anneal 30s, 72 DEG C
Extend 20s, circulate 4 times；Last 72 DEG C of extensions 10min.The filling-in product for obtaining as Araneus ventricosus dragline silk protein list
The double-stranded sequence of body.
Embodiment 2 builds pSIMPLE-19 EcoR V/BAP monomer recombinant plasmids
1st, the double-stranded sequence of above-mentioned filling-in product Araneus ventricosus dragline silk protein monomer is taken into 5uL carries out Ago-Gel electricity
Swimming, is tried using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 glue reclaims of Takara
Agent box carries out rubber tapping recovery to purpose band 126bp.
2nd, the filling-in product of above-mentioned recovery is carried out into phosphatizing treatment：
Reaction system is：
ddH2O 10.25 μl、
10×T4 DNA Polynucleotide Kinase Buffer 2.5 μl、
The μ l of filling-in product 12 of above-mentioned recovery,
ATP 25 nmol (0.25 μl) 、
T4 Polynucleotide Kinase 10 units。
Reaction condition is：Reacted 30 minutes at 37 DEG C.
3rd, the filling-in product of phosphorylation and pSIMPLE-19 EcoR V/BAP expression vectors are attached again：
Reaction system is：
pSIMPLE-19 EcoR V/BAP Vector 1 μl
The μ l of Insert DNA (above-mentioned Phosphorylated products) 4
Solution I (DNA Ligation Kit Ver.2.1) 5 μl
Reaction condition is：2h is reacted at 16 DEG C.
4th, connection product is transformed into bacillus coli DH 5 alpha competent cell, is containing Amp（100 µg/mL）Selection
80 μ l bacterium solutions are coated with mild-natured plate, 15~18 h are cultivated, picking white colony is inoculated into 800uL LB fluid nutrient mediums（Contain
100 µg/mL Amp）, 37 DEG C, more than the h of shaking table shaken cultivation 6；Take 2 μ L bacterium solutions and do template, enter performing PCR identification.
The PCR reaction volumes are 25 μ L, Premix Taq enzymes, universal primer M13F and M13R（Sequence is respectively such as SEQ
Shown in ID NO.3 and SEQ ID NO.4）.The PCR reaction conditions are：94 DEG C of predegeneration 1min；94 DEG C of denaturation 25s, 49 DEG C are moved back
Fiery 25s, 72 DEG C of extension 30s, circulates 32 times；Last 72 DEG C of extensions 10min.
Result is as shown in figure 4, have 3 clones to have a bright purpose band at 250bp or so places, and send Hua Da
The further sequencing identification of genome company, sequence is completely correct, successfully obtains pSIMPLE-19 EcoR V/BAP monoploid restructuring matter
Grain.
Embodiment 3 builds Araneus ventricosus many times of concatermers of dragline silk protein core repeat sequence
1st, culture conversion has the bacterium solution of pSIMPLE-19 EcoR V/BAP monoploid recombinant plasmids, using the small extraction reagent kit of plasmid
（TIANprep Mini Plasmid Kit）Extracting plasmid.
By plasmid restriction enzyme Nhe I/Hind III double digestions, reaction composition and step are as follows：
The μ g of 1 × plasmids of pSIMPLE-19 2
10 × Quickcut buffer 5μL
Nhe I 1μL
Hind Ⅲ 1μL
dd H2O is mended to 50 μ L.
Above-mentioned reaction system gently mix after brief centrifugation, 37 DEG C insulation 2h.
2nd, separated with 1% agarose gel electrophoresis, analyze endonuclease bamhi size（See Fig. 5）, with DNA glue reclaim kits
（TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0）Reclaim genes of interest fragment；Again will
Monomer recombinant plasmid restriction enzyme Spe I/Hind III double digestions, reaction composition and step are as follows：
The μ g of 1 × plasmids of pSIMPLE-19 2
10 × Quickcut buffer 5μL
Spe I 1μL
HindⅢ 1μL
dd H2O is mended to 50 μ L
Above-mentioned reaction system gently mix after brief centrifugation, 37 DEG C insulation 2h.
3rd, separated with 1% agarose gel electrophoresis, analyze endonuclease bamhi size（See Fig. 5）, with DNA glue reclaim kits
（TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0）Reclaim linear carrier.By what is reclaimed
Genes of interest fragment and linear carrier are attached by proper proportion with T4 DNA ligases, and reaction system is as follows：
The μ L of genes of interest fragment 6
The μ L of linear carrier 2
10 × T4 ligase buffer 1μL
T4 ligase 1μL
Coupled reaction 8h at 16 DEG C.
4th, enzyme connect product thing is converted into bacillus coli DH 5 alpha competent cell, positive recombinant is screened with bacterium solution PCR methods
（Fig. 6）, it is seen that 3 clones have a bright purpose band at 350bp or so places, and send Huada gene company be sequenced into
The identification of one step, successfully obtains 2 times of concatermers of Araneus ventricosus dragline silk protein core repeat sequence.
5th, repeat the above steps, successively obtained four times of concatermers, octuple concatermer and 16 times of concatermers.
Fig. 7 is the PCR qualification figures of 4 times of concatermer recombinant plasmid clones, and 8 clones have the molecular weight to be
The purpose band of 578bp, is all positive colony.
Fig. 8 is the PCR qualification figures of 8 times of concatermer recombinant plasmid clones, and 5 clones have the molecular weight to be
The purpose band of 1034bp, is all positive colony.
Fig. 9 is the PCR qualification figures of 16 times of concatermer recombinant plasmid clones, and 3 clones have the molecular weight to be
The purpose band of 1936bp, therefore have 3 in 4 clones identified for positive colony.This includes for the 1936bp of purpose band
16 times of concatermer sequences（1836bp）, primer length and with a distance from purpose fragment both sides, common 1936bp.
So far, have successfully been obtained the plan Araneus ventricosus dragline silk protein of 1836bp（As shown in SEQ ID NO.5, figure
10）.
Embodiment 4 intends the expression of the coded sequence of Araneus ventricosus dragline silk protein
Verified by lot of experiments, the plan Araneus ventricosus dragline silk protein of above-mentioned structure can successful translation give expression to big abdomen
Epeira draws silk-fibroin.The following is a kind of prokaryotic expression system：
The biology plasmid of the plan Araneus ventricosus dragline silk protein with 1836bp that will be successfully obtained, respectively with Nhe I and
III two kinds of restriction endonuclease of Hind carry out digestion, reclaim digestion products, are reacted by T4 DNA ligases, will reclaim
Digestion products be connected with linearized p ET28a (+) plasmid, construct the original containing spider silk core sequence concatermer
Nuclear expression carrier.
Prokaryotic expression carrier is converted into BL21 (DE3) competent cell again, incubated overnight, picking monoclonal is laggard
Row activation and amplification culture.
As a result：Successfully be detected the traction silk-fibroin for intending going out expressed by Araneus ventricosus dragline silk protein sequence.
SEQUENCE LISTING
<110>Zhongshan University
<120>A kind of artificial constructed Araneus ventricosus dragline silk protein and its construction method
<130>
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 108
<212> DNA
<213>Araneus ventricosus draw silk-fibroin cDNA core repeat sequences
<400> 1
gccgcggcag ccgcagcagc agctggtgga caaggaggtc aaggtggata tggaggatta 60
ggttcccaag gagctggaca aggaggatat ggagcaggac aaggtggt 108
<210> 2
<211> 126
<212> DNA
<213>The sequence monomer of Araneus ventricosus dragline silk protein
<400> 2
gctagcgccg cggcagccgc agcagcagct ggtggacaag gaggtcaagg tggatatgga 60
ggattaggtt cccaaggagc tggacaagga ggatatggag caggacaagg tggtactagt 120
aagctt 126
<210> 3
<211> 18
<212> DNA
<213>The sequence of universal primer M13F
<400> 3
tgtaaaacga cggccagt 18
<210> 4
<211> 18
<212> DNA
<213>The sequence of universal primer M13R
<400> 4
caggaaacag ctatgacc 18
<210> 5
<211> 1836
<212> DNA
<213>Artificial constructed plan Araneus ventricosus dragline silk protein sequence
<400> 5
gctagcgccg cggcagccgc agcagcagct ggtggacaag gaggtcaagg tggatatgga 60
ggattaggtt cccaaggagc tggacaagga ggatatggag caggacaagg tggtactagc 120
gccgcggcag ccgcagcagc agctggtgga caaggaggtc aaggtggata tggaggatta 180
ggttcccaag gagctggaca aggaggatat ggagcaggac aaggtggtac tagcgccgcg 240
gcagccgcag cagcagctgg tggacaagga ggtcaaggtg gatatggagg attaggttcc 300
caaggagctg gacaaggagg atatggagca ggacaaggtg gtactagcgc cgcggcagcc 360
gcagcagcag ctggtggaca aggaggtcaa ggtggatatg gaggattagg ttcccaagga 420
gctggacaag gaggatatgg agcaggacaa ggtggtacta gcgccgcggc agccgcagca 480
gcagctggtg gacaaggagg tcaaggtgga tatggaggat taggttccca aggagctgga 540
caaggaggat atggagcagg acaaggtggt actagcgccg cggcagccgc agcagcagct 600
ggtggacaag gaggtcaagg tggatatgga ggattaggtt cccaaggagc tggacaagga 660
ggatatggag caggacaagg tggtactagc gccgcggcag ccgcagcagc agctggtgga 720
caaggaggtc aaggtggata tggaggatta ggttcccaag gagctggaca aggaggatat 780
ggagcaggac aaggtggtac tagcgccgcg gcagccgcag cagcagctgg tggacaagga 840
ggtcaaggtg gatatggagg attaggttcc caaggagctg gacaaggagg atatggagca 900
ggacaaggtg gtactagcgc cgcggcagcc gcagcagcag ctggtggaca aggaggtcaa 960
ggtggatatg gaggattagg ttcccaagga gctggacaag gaggatatgg agcaggacaa 1020
ggtggtacta gcgccgcggc agccgcagca gcagctggtg gacaaggagg tcaaggtgga 1080
tatggaggat taggttccca aggagctgga caaggaggat atggagcagg acaaggtggt 1140
actagcgccg cggcagccgc agcagcagct ggtggacaag gaggtcaagg tggatatgga 1200
ggattaggtt cccaaggagc tggacaagga ggatatggag caggacaagg tggtactagc 1260
gccgcggcag ccgcagcagc agctggtgga caaggaggtc aaggtggata tggaggatta 1320
ggttcccaag gagctggaca aggaggatat ggagcaggac aaggtggtac tagcgccgcg 1380
gcagccgcag cagcagctgg tggacaagga ggtcaaggtg gatatggagg attaggttcc 1440
caaggagctg gacaaggagg atatggagca ggacaaggtg gtactagcgc cgcggcagcc 1500
gcagcagcag ctggtggaca aggaggtcaa ggtggatatg gaggattagg ttcccaagga 1560
gctggacaag gaggatatgg agcaggacaa ggtggtacta gcgccgcggc agccgcagca 1620
gcagctggtg gacaaggagg tcaaggtgga tatggaggat taggttccca aggagctgga 1680
caaggaggat atggagcagg acaaggtggt actagcgccg cggcagccgc agcagcagct 1740
ggtggacaag gaggtcaagg tggatatgga ggattaggtt cccaaggagc tggacaagga 1800
ggatatggag caggacaagg tggtactagt aagctt 1836
Claims (10)
1. a kind of sequence monomer of Araneus ventricosus dragline silk protein, it is characterised in that its sequence such as SEQ ID NO.2 institutes
Show.
2. it is a kind of to intend Araneus ventricosus dragline silk protein, it is characterised in that its sequence is as shown in SEQ ID NO.5.
3. Araneus ventricosus dragline silk protein answering in terms of production Araneus ventricosus traction silk-fibroin is intended described in claim 2
With.
4. a kind of artificial constructed Araneus ventricosus draw silk-fibroin, it is characterised in that the sequential coding as shown in SEQ ID NO.5 is turned over
Translate and obtain.
5.SEQ ID NO.1, sequence shown in SEQ ID NO.2 or SEQ ID NO.5 are in artificial constructed Araneus ventricosus traction fiber egg
Application in terms of white gene.
6. a kind of method of artificial constructed Araneus ventricosus dragline silk protein, it is characterised in that comprise the following steps：
（1）Sequence shown in SEQ ID NO.2 is divided into 2 ends 18 72bp fragments of base complementrity, and synthesis obtains two
Oligonucleotide chain, by complementation connection filling-in, obtains double-stranded sequence；The double-stranded sequence is connected to pSIMPLE-19 EcoR
V/BAP expression vectors, obtain pSIMPLE-19 EcoR V/BAP monomer recombinant plasmids；
（2）Recombinant plasmid is respectively through Nhe I/Hind III double digestions and Spe I/Hind III double digestions, two digestions
Product is connected, and obtains 2 times of concatermers of Araneus ventricosus dragline silk protein core repeat sequence；The step is repeated, big abdomen is obtained
16 times of concatermers of epeira dragline silk protein core repeat sequence, as artificial constructed Araneus ventricosus draw the volume of silk-fibroin
Code sequence, as shown in SEQ ID NO.5.
7. method according to claim 5, it is characterised in that comprise the following steps：
S1. the sequence monomer of Araneus ventricosus dragline silk protein is prepared
S11. according to Araneus ventricosus macromolecule traction fiber protein part cDNA sequence, core repeat sequence, such as SEQ ID are determined
Shown in NO.1；
S12. 5 of core repeat sequence shown in SEQ ID NO.1’End adds Nhe I restriction enzyme sites, 3’End adds Spe I enzymes
Enzyme site and Hind III digestions site, obtain the sequence monomer of Araneus ventricosus dragline silk protein, such as SEQ ID NO.2 institutes
Show；
S13. gene monomer sequence shown in above-mentioned SEQ ID NO.2 is divided into 2 fragments of 72bp, the end of two fragments has
18 base complementrities；Sequent synthesis according to two fragments obtain two oligonucleotide chains；
S14. two synthetic oligonucleotide chains are carried out into complementary connection, and filling-in is carried out by PCR reactions, obtain 2 ends
The double-stranded sequence of the Araneus ventricosus dragline silk protein monomer with restriction enzyme site；
S2. pSIMPLE-19 EcoR V/BAP monomer recombinant plasmids are built
S21. the double-stranded sequence of above-mentioned Araneus ventricosus dragline silk protein monomer is carried out into phosphorylation；
S22. the product of phosphorylation is attached with pSIMPLE-19 EcoR V/BAP expression vectors again；
S23. by connection product transformed competence colibacillus cell, by the screening and identification of transformant, pSIMPLE-19 EcoR are obtained
V/BAP monomer recombinant plasmids；
S3. Araneus ventricosus many times of concatermers of dragline silk protein core repeat sequence are built
S31. culture conversion has the bacterium solution of pSIMPLE-19 EcoR V/BAP monomer recombinant plasmids, extracts pSIMPLE-19
EcoR V/BAP monomer recombinant plasmids；
S32. pSIMPLE-19 EcoR V/BAP monomers recombinant plasmid is used into restriction enzyme Nhe I/Hind respectively
III double digestions, with restriction enzyme Spe I/Hind III double digestions, be separately recovered and obtain genes of interest and linear plasmid
Carrier；
S33. the genes of interest fragment and linear plasmid carrier of recovery are attached；Connection product transformed competence colibacillus cell；
S34. positive recombinant is screened with bacterium solution PCR methods, so as to obtain Araneus ventricosus dragline silk protein core repeat sequence
2 times of concatermers；
S35. Araneus ventricosus dragline silk protein 2 times of concatermer plasmids of core repeat sequence are extracted, above-mentioned S32-S34 is repeated
Method, obtain Araneus ventricosus dragline silk protein 16 times of concatermers of core repeat sequence, as artificial constructed great Fu gardens
Spider draws the coded sequence of silk-fibroin, as shown in SEQ ID NO.5.
8. method according to claim 6, it is characterised in that the reaction volume of PCR reactions is 25 μ L described in step S14,
Reaction system is included：5 × PrimeSTAR buffer solutions, 200 μM of dNTPs, 0.75 U PrimeSTAR HS DNA polymerases；
PCR reaction conditions are described in step S14：94 DEG C of predegeneration 2min；94 DEG C of denaturation 1min, 49 DEG C of annealing 30s, 72 DEG C of extension 20s,
Circulation 4 times；Last 72 DEG C of extensions 10min.
9. method according to claim 6, it is characterised in that the method for phosphorylation is described in step S21：Take Araneus ventricosus
The double-stranded sequence of dragline silk protein monomer enters row agarose gel electrophoresis, and the purpose band to 126bp carries out rubber tapping recovery
Afterwards, phosphatizing treatment is carried out；The reaction system of phosphorylation is：ddH2O 10.25μl、10×T4 DNA Polynucleotide
The μ l of Kinase Buffer 2.5, the μ l of filling-in product 12 of above-mentioned recovery, ATP 25nmol 0.25 μ l, T4
Polynucleotide Kinase 10units；The reaction condition of phosphorylation is：Reacted 30 minutes at 37 DEG C.
10. method according to claim 5, it is characterised in that the reaction system connected described in step S22 is：
The μ l of pSIMPLE-19 EcoR V/BAP expression vectors 1, the μ l of above-mentioned Phosphorylated products 4, the μ l of Solution I 5；The reaction of connection
Condition is：2h is reacted at 16 DEG C；The reaction system of double digestion is described in step S32：The μ g of plasmid 2, the μ L of reaction buffer 5, inscribe
Each 1 μ L, ddH of enzyme2O is mended to 50 μ L；The reaction condition of double digestion is described in step S32：Reaction system after gently mixing it is instantaneous from
The heart, 37 DEG C of insulation 2h.
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| CN107365791A (en) * | 2017-08-23 | 2017-11-21 | 中山大学 | A kind of new method of vivoexpression Araneus ventricosus traction silk-fibroin |
| CN110283243A (en) * | 2019-06-10 | 2019-09-27 | 华南师范大学 | Araneus ventricosus traction fiber performance and its Silk gene sequence |
| CN110317252A (en) * | 2019-06-10 | 2019-10-11 | 华南师范大学 | Clouding spider traction fiber performance and its Silk gene sequence |
| CN113789341A (en) * | 2021-09-16 | 2021-12-14 | 陕西理工大学 | Homotype tandem polymer of BmSPI38, and construction method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365791A (en) * | 2017-08-23 | 2017-11-21 | 中山大学 | A kind of new method of vivoexpression Araneus ventricosus traction silk-fibroin |
| CN110283243A (en) * | 2019-06-10 | 2019-09-27 | 华南师范大学 | Araneus ventricosus traction fiber performance and its Silk gene sequence |
| CN110317252A (en) * | 2019-06-10 | 2019-10-11 | 华南师范大学 | Clouding spider traction fiber performance and its Silk gene sequence |
| CN113789341A (en) * | 2021-09-16 | 2021-12-14 | 陕西理工大学 | Homotype tandem polymer of BmSPI38, and construction method and application thereof |
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