CN110776569B

CN110776569B - Diblock fusion protein with adhesion-freeze resistance dual functions and synthesis method and application thereof

Info

Publication number: CN110776569B
Application number: CN201910951884.1A
Authority: CN
Inventors: 张雷; 杨静; 张相宇; 高弈航
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2019-10-09
Filing date: 2019-10-09
Publication date: 2022-03-15
Anticipated expiration: 2039-10-09
Also published as: CN110776569A

Abstract

The invention relates to a diblock fusion protein with adhesion-freeze resistance dual functions, a synthetic method and application thereof; the gene expression vector containing both antifreeze protein gene and adhesion protein gene is prepared by using gene engineering as technological means, transformed into host cell, over-expressed by inducer, separated and purified to obtain fusion protein. The amino acid sequence of the fusion protein is SEQ ID NO.3, and the nucleotide sequence is SEQ ID NO. 4. Mussel adhesive protein and antifreeze protein are fused at gene level, thus constructing diblock fusion protein with double functions of adhesion and antifreeze; the fusion protein is expressed by utilizing escherichia coli fermentation, the supernatant protein is purified by utilizing an affinity chromatography technology, the inclusion body is purified by utilizing acetic acid, a high-purity separation and purification method is constructed, the fusion protein is synthesized by a microbial one-step method, the process is mild and efficient, the cost is low, the equipment requirement is low, and the later-stage expanded production is easy to realize.

Description

Diblock fusion protein with adhesion-freeze resistance dual functions and synthesis method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a diblock fusion protein with adhesion-freeze resistance dual functions, a synthetic method and application thereof; comprises the gene design of diblock fusion protein with adhesion-antifreeze double functions, the construction of a recombinant expression vector containing a fusion protein gene segment, the fermentation expression in escherichia coli, the separation and purification method of the fusion protein and the application thereof in the aspect of antifreeze surface.

Background

Antifreeze proteins (AFPs) are a general term for a class of proteinaceous compounds with enhanced biological antifreeze capability, which bind to small ice crystals and prevent the crystallization of ice and the growth of crystals. In general, the greater the thermal hysteresis value of antifreeze proteins, the better the adaptability of the organism to low temperatures. (thermal hysteresis is the lowering of the freezing point of a solution in a non-linear manner without changing its melting point, thereby creating a difference between the freezing point and the melting point of the solution, the difference between the melting point and the melting point of the solution being referred to as the thermal hysteresis value.)

The antifreeze proteins are classified into antifreeze proteins and freeze-resistant proteins. Antifreeze-like proteins can protect their body fluids from freezing together, but in extremely cold environments AFP function is impaired, leading to rapid growth of ice crystals, leading to death of the organism; the antifreeze proteins are believed to act as cryoprotectants to protect organisms from freezing, rather than freezing, so that species containing the antifreeze proteins survive freezing of body fluids.

To date, a number of known classes of AFPs have been discovered, including fish AFPs, plant AFPs, insect AFPs, and AFPs of marine ice microbial species. Among them, fish AFP is most clearly studied and classified into antifreeze glycoprotein, I-type AFP, II-type AFP, III-type AFP, and IV-type AFP. Antifreeze glycoproteins (AFGP) are polymers composed of a glycine-threonine tripeptide repeat unit, in which threonine is attached to a disaccharide group. Type I AFP is rich in alanine (over 60%) and has a regular alpha-helical structure. Type II AFP contains a high content of cysteine (8%) and has a structure comprising an alpha-helical structure, a beta-sheet structure and a large number of random structures. Type III AFP is a small globular protein (62-66 amino acids) with a flat ice binding surface. In type IV AFP 4 α -helical structures are arranged antiparallel. Research on plant AFP and insect AFP has shown that most of them have beta sheet structures that can bind well to basal planes and facets of ice, making these proteins highly active. Such proteins are referred to as "high antifreeze proteins". Compared with the common antifreeze protein, the high-activity antifreeze protein can be adsorbed on the edge surface of the ice crystal and also can be adsorbed on the basal plane of the ice crystal. The characteristics of the antifreeze protein enable the antifreeze protein to have wide application prospect in the fields of industry, agriculture, medicine and the like. For example, it is used as antifreezing agent in industry, and can be used for introducing antifreezing gene into DNA sequence of crops by means of transgenic technology, prolonging its production season, or medically preserving cells and organs at low temp.

However, purification and preparation of antifreeze proteins is very difficult, thus greatly limiting their applications. The hydrolysate of the protein is a polypeptide. The polypeptide synthesis technology is mature, such as solid phase synthesis method, liquid phase synthesis method, acidolysis method, alkaline hydrolysis method, enzyme method and the like. Therefore, the polypeptide with antifreeze activity is designed and synthesized, so that the polypeptide has the effect similar to that of antifreeze protein, and the application of the polypeptide in various fields is favorably expanded, and the large-scale industrial production is realized.

So far, the research on anti-icing materials mainly focuses on utilizing hydrophobic or super-hydrophobic surfaces, but the anti-icing effect of hydrophobic materials is not ideal, and the preparation method of super-hydrophobic materials is complicated. A few scientists also modify the antifreeze protein and the polymer on the surface of the material to obtain a good anti-icing effect, but the antifreeze protein is combined with the polymer, a reactive group needs to be modified on the protein, the protein is easy to inactivate, and the operation is complicated.

Disclosure of Invention

The invention takes gene engineering as a technical means to prepare a gene expression vector containing an antifreeze protein gene and an adhesion protein gene at the same time, transforms the gene expression vector into a host cell, excessively expresses the protein through an inducer, and separates and purifies to obtain the fusion protein. The anti-freezing surface effect prepared from the diblock fusion protein with the adhesion-anti-freezing dual function is obvious, and the preparation method is simple and easy to implement, does not need special reaction conditions and has wide application range.

The first purpose of the invention is to obtain a diblock fusion protein with specific adhesion and anti-freezing functions by using a genetic engineering technology. Wherein, the first block uses mussel byssus protein as an anchoring functional unit, and the second block uses antifreeze protein as an antifreeze functional unit.

In order to achieve the purpose, the technical scheme provided by the invention is as follows: a fusion protein with specific adhesion and anti-freezing functions has an amino acid sequence shown in SEQ ID No. 3.

The mussel adhesive protein amino acid sequence of the fusion protein comprises a sequence shown by SEQ ID NO.1, and the antifreeze protein amino acid sequence comprises a sequence shown by SEQ ID NO. 2.

The second purpose of the invention is to provide the synthesis method of the fusion protein, which is characterized in that the gene of the fusion protein is cloned to a plasmid vector to obtain a recombinant expression plasmid, the recombinant expression plasmid and the plasmid expressing tyrosinase are transformed into a host cell together, then the host cell is induced by an inducer to excessively express the fusion protein, and the fusion protein is obtained after separation and purification.

The technical scheme is as follows:

a diblock fusion protein with adhesion-antifreeze double functions is characterized in that the fusion protein contains mussel adhesion protein and antifreeze protein.

The mussel adhesive protein comprises a sequence shown in SEQ ID NO. 1.

The antifreeze protein comprises a sequence shown in SEQ ID NO. 2.

The invention relates to a method for synthesizing diblock fusion protein with adhesion-freeze resistance dual functions, which is characterized by comprising the following steps: cloning the gene of the fusion protein to a plasmid vector to obtain a recombinant expression plasmid, transferring the recombinant expression plasmid and the plasmid expressing tyrosinase into host cells together, inducing the host cells to excessively express the fusion protein by using an inducer, and separating and purifying to obtain the fusion protein.

Preferably, the amino acid sequence of the fusion protein is SEQ ID NO.3, and the nucleotide sequence is SEQ ID NO. 4. The recombinant expression vector is obtained by cloning diblock fusion protein genes into an escherichia coli expression vector pET-28a, and contains BamHI and NotI restriction enzyme sites, a strong T7 promoter, a lca lactose operon and resistance screening genes of kanamycin and chloramphenicol.

The host cell for expressing the fusion protein is the host cell containing the nucleotide sequence of SEQ ID NO. 4; or a host cell containing a recombinant expression vector. Preferably, the cell is Escherichia coli BL21(DE 3).

Preferably, the synthesis method of the fusion protein comprises the steps of fusing the mussel adhesive protein and the antifreeze protein by using a genetic engineering technology to construct a recombinant expression vector, transforming the recombinant expression vector and an expression vector for expressing tyrosinase into escherichia coli BL21(DE3) together to express the fusion protein, and purifying the mussel adhesive protein/antifreeze protein fusion protein by using a column chromatography or acetic acid extraction method. The method comprises the following steps:

(1) artificially synthesizing a diblock fusion protein gene, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 4;

(2) cloning the synthesized diblock fusion protein gene into an escherichia coli expression vector pET-28a, adding a BamHI restriction enzyme site at the 5 'end of the fusion protein gene, and adding a Not I restriction enzyme site at the 3' end of the gene to obtain a recombinant expression vector (shown in figure 1);

(3) constructing an expression vector for expressing tyrosinase (as shown in figure 2);

(4) the successfully constructed recombinant expression vector and the expression vector for expressing tyrosinase are jointly transformed into escherichia coli BL21(DE3), and LB solid medium (10 g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl and 15g/L of agar) containing kanamycin and chloramphenicol double resistance is utilized; screening transformants by using a plate;

(5) escherichia coli BL21(DE3) is fermented to express diblock fusion protein SEQ ID No.3 with adhesion-freeze resistance dual functions; the nucleotide sequence is SEQ ID NO. 4;

(6) and (3) separating and purifying the diblock fusion protein.

The host cell for expressing the fusion protein is a host cell containing the nucleotide sequence of SEQ ID NO.4 or a host cell containing a recombinant expression vector;

the host cell is Escherichia coli BL21(DE 3); the recombinant expression vector is obtained by cloning the fusion protein gene into an escherichia coli expression vector pET-28a, and contains BamH I and Not I restriction enzyme sites, a T7 strong promoter, a lca lactose operon, a hexahistidine tag and resistance screening genes of kanamycin and chloramphenicol.

Compared with the prior art, the invention has the beneficial effects that: in the invention, the mussel adhesive protein and the antifreeze protein are fused at the gene level for the first time, so that the diblock fusion protein with the double functions of adhesion and antifreeze is constructed. The fusion protein is expressed by utilizing escherichia coli fermentation, the supernatant protein is purified by utilizing an affinity chromatography technology according to the unique property of the fusion protein, the inclusion body is purified by utilizing acetic acid, a high-purity separation and purification method is constructed, the fusion protein is synthesized by a microbial one-step method, the process is mild and efficient, the cost is low, the equipment requirement is low, and the later-stage expanded production is easy. Partial conversion of tyrosine residues in fusion proteins in E.coli to the unnatural amino acid DOPA was achieved using tyrosinase. The mussel adhesive protein part of the hydroxylated fusion protein contains DOPA, and the fusion protein can be firmly adhered to the surface of a matrix material through covalent bonds and coordination capacity to form a surface protein modification layer; meanwhile, the antifreeze protein has no toxic influence on the growth of normal animal cells, and is expected to become an antifreeze coating material for medical devices and biochips.

Drawings

FIG. 1: schematic diagram of recombinant expression vector pET-28 a-MP-AFP;

the recombinant expression vector contains BamHI and NotI restriction sites, a strong T7 promoter, a lca lactose operon, a hexahistidine tag and resistance screening genes of kanamycin and chloramphenicol.

FIG. 2: a schematic representation of a recombinant expression vector for expressing tyrosinase;

FIG. 3: double enzyme cutting of the recombinant strain;

FIG. 4: a comparison graph of unhydroxylated mussel byssus protein and MP-AFP;

FIG. 5: and (3) testing the freezing resistance of the fusion protein.

Detailed Description

The invention provides a preferable diblock fusion protein MP-AFP (named MP-AFP (polypeptide injection foot protein-antidireze protein)) with double functions of adhesion and freeze resistance, and the amino acid sequence of the protein MP-AFP is shown as SEQ ID NO. 3. The nucleotide sequence is shown in SEQ ID NO. 4.

The fusion protein MP-AFP gene of the invention is subjected to codon optimization according to the expression preference of escherichia coli, so that the fusion protein can be stably expressed in the escherichia coli in an excessive way.

The invention provides a recombinant expression vector containing the fusion protein MP-AFP gene, which is characterized in that the mussel adhesive protein/antifreeze protein fusion protein MP-AFP gene is cloned into an escherichia coli expression vector pET-28a to obtain a recombinant expression vector pET-28a-MP-AFP (which is named as pET-28a-MP-AFP), wherein the recombinant expression vector contains BamH I and Not I restriction enzyme cutting sites, a strong T7 promoter, a lca lactose operon, a hexahistidine tag and resistance screening genes of kanamycin and chloramphenicol. The invention provides a host cell containing the fusion protein MP-AFP gene, which is characterized in that the host cell is a host cell containing a nucleotide sequence shown as SEQ ID NO.3 or a host cell containing the recombinant expression vector pET-28a-MP-AFP, and preferably, the host cell is escherichia coli BL21(DE 3).

The invention provides a method for producing the fusion protein MP-AFP, which is characterized in that mussel adhesive protein MP and KE zwitterionic polypeptide are fused by using a genetic engineering technology to construct a recombinant expression vector pET-28a-MP-AFP, then pET-28a-MP-AFP plasmid vector is transformed into escherichia coli BL21(DE3) to express the fusion protein MP-AFP, and the mussel diblock fusion protein MP-AFP is purified by a column chromatography or acetic acid extraction method

Example 1: construction of recombinant expression vectors and strains

Construction of recombinant expression vector

3 GGGGS repetitive sequences are used as connecting polypeptides to connect adhesion protein and mealworm antifreeze protein to form fusion protein MP-AFP, and the gene is subjected to codon optimization according to the expression preference of escherichia coli on the basis of not changing the amino acid sequence of the fusion protein MP-AFP. Adding a BamH I restriction site at the 5 'end of the gene sequence and a Not I restriction site at the 3' end of the gene sequence; the gene sequence is artificially synthesized and cloned into an escherichia coli expression vector pET-28a to obtain a recombinant expression vector pET-28a-MP-AFP, wherein the recombinant expression vector contains a strong promoter of T7, a lca lactose operon, a kanamycin resistance marker site and a hexahistidine tag, and is shown in figure 1.

② chemical transformation of Escherichia coli

Competent E.coli BL21(DE3) was thawed on ice. Uniformly mixing a correctly constructed recombinant expression vector pET-28a-MK-AFP, an expression vector for expressing tyrosinase and competent escherichia coli BL21(DE3), incubating on ice for 30min, thermally shocking at 42 ℃ for 45s, and rapidly standing on ice for 3 min; adding 450 mul LB culture medium, shaking for resuscitation for 2h, taking 100 mul bacterial liquid after resuscitation, evenly coating on a double-resistance LB solid culture medium plate containing kanamycin and chloramphenicol for resistance screening; after the same single colony is picked out for overnight culture by aseptic technique, the plasmid of the escherichia coli is extracted according to a plasmid miniprep kit, the extracted plasmid is cut by BamH I restriction enzyme and Not I restriction enzyme and is subjected to agarose electrophoresis, and two bands are cut by the plasmid, wherein the two bands are respectively about 700bp and 5.5kb (as shown in figure 3). The sizes of the fusion protein gene and the pET-28a vector respectively accord with the sizes of the fusion protein gene and the pET-28a vector which are theoretically obtained. Simultaneously, the extracted plasmids are sent to Jinweinzhi company for sequencing; comparing the sequencing result with the gene sequence shown in SEQ ID NO.4, wherein the similarity is more than 99%, and the gene fragment is considered as the fusion protein MP-AFP gene. And the colony which is successfully subjected to colony PCR, plasmid double digestion and sequencing is taken as a target transformant. Successfully transformed E.coli strains were stored in glycerol at-80 ℃.

Example 2: expression and purification of fusion protein MP-AFP

The colibacillus fermentation expression fusion protein MP-AFP

Successfully transformed E.coli was streaked and activated on double-resistant LB solid medium plates containing kanamycin and chloramphenicol. Single colonies were picked up in a 5ml tube containing 50. mu.g/ml of LB liquid medium containing kanamycin and chloramphenicol, and shake-cultured overnight at 37 ℃ at 200 rpm. Transferring the Escherichia coli seed liquid into a 500ml shake flask containing 200ml of culture medium according to the proportion of 1: 100; measuring the growth curve of Escherichia coli by using an ultraviolet spectrophotometer, and after the Escherichia coli is transferred, determining the concentration of the bacterial liquid to be OD₆₀₀When the strain is equal to 0.8, 0.8mM IPTG is used for inducing the Escherichia coli to express the fusion protein MP-AFP, and the Escherichia coli is collected after further culturing for 12h under the conditions of 37 ℃ and 250 rpm.

② separation and purification of fusion protein MP-AFP

The Escherichia coli fermentation broth is centrifuged at 6000rpm at 4 ℃ for 10min, and the culture medium is discarded. Resuspending Escherichia coli thallus with PBS solution, ultrasonically crushing the bacteria liquid in ice water bath for 40min, outputting power of 200W, crushing for 2s, and stopping for 3 s; and (2) centrifuging the crushed clarified bacterial liquid at 4 ℃ to respectively obtain a supernatant and an inclusion body, adding the escherichia coli thallus, the crushed supernatant and the crushed inclusion body into a loading buffer solution (loading buffer), boiling, and then carrying out polyacrylamide gel (SDS-PAGE) electrophoresis to obtain the fusion protein MP-AFP, so that the fusion protein MP-AFP in the supernatant is separated by an affinity chromatography method in a subsequent experiment, and the fusion protein MP-AFP is separated and purified from the inclusion body by acetic acid.

a. Separation and purification of supernatant of MK-AFP:

after the fermentation of the Escherichia coli is finished, the strain is centrifuged for 10min at the temperature of 4 ℃ and the rpm of 6000 to collect the strain. The Escherichia coli cells were resuspended in binding buffer (20mM sodium phosphate, 0.5M sodium chloride, 40mM imidazole, pH7.4), and the cells were sonicated in an ice water bath for 40min, with an output of 200W for 2s and 3s rest. The clear bacterial liquid obtained by crushing is centrifuged at 4 ℃ to obtain the supernatant. The supernatant containing the fusion protein MP-AFP collected after centrifugation was filtered through a 0.22 μm membrane to remove solid particles. 5ml Histrap column from GE company is selected to carry out affinity chromatography separation on supernatant of fusion protein MP-AFP. The Histrap column was ligated to AKAT prime plus. The Histrap column was washed sequentially with 10 column volumes of high purity water and binding buffer until the baseline leveled off. And (3) feeding the supernatant of the fusion protein MP-AFP passing through the membrane into the column at the flow rate of 1ml/min, so that the fusion protein MP-AFP is bonded on the column. After the end of the loading, the column was washed with a wash buffer (20mM sodium phosphate, 0.5M sodium chloride, 320mM imidazole, pH7.4) at a flow rate of 2ml/min to bring the base line to a flat state. After the washing, the fusion protein MP-AFP bound to the column was eluted with an elution buffer (20mM sodium phosphate, 0.5M sodium chloride, 500mM imidazole, pH7.4) and the eluate at the time of the appearance of the absorption peak was collected. The Histrap column was washed with binding buffer and high purity water in sequence at 8 column volumes, and finally the Histrap column was stored in 20% ethanol. The eluent is ultrafiltered by an ultrafiltration tube while the liquid is changed by high-purity water. The resulting solution was lyophilized with a lyophilizer.

b. Separation and purification of the fusion protein MP-AFP precipitate:

the obtained inclusion bodies were washed 2 times for 30min each with an inclusion body washing solution containing 2M urea, 0.6% Triton-X-100, 50mM PBS, 4mM EDTA to remove foreign proteins other than the inclusion bodies. 60% acetic acid heavy suspension inclusion body, at 25 degrees C, 250rpm conditions under shaking treatment for 3h, make most inclusion body protein dissolved in the acid solution. The treated acetic acid supernatant was dialyzed overnight at 4 ℃ for 18 h. After the dialysis is finished, the solution in the dialysis bag is centrifuged at 4 ℃, then is ultrafiltered and concentrated by an ultrafiltration tube, and is freeze-dried by a freeze dryer to obtain the fusion protein MP-AFP.

Dissolving the purified fusion protein MP-AFP in a PBS buffer solution, adding a loading buffer solution (loading buffer), boiling, performing polyacrylamide gel electrophoresis, and analyzing the purity, wherein the purity of the purified fusion protein is more than 80%, and the molecular weight of the purified fusion protein is about 36 KD.

Example 3: surface modification and atomic force microscopy analysis

Surface modification of matrix material by sample

Glass sheets and mica sheets are used as matrix materials, and water and fusion protein MP-AFP are used as samples to modify the surface of the matrix materials. The cleaned matrix material was soaked in both samples and incubated at 25 ℃ at 80% humidity for 12h, at least 3 replicates per group. After the incubation was completed, the matrix material was washed 3 times with high-purity water to remove the excess protein sample.

② atomic force microscope detection

The surface appearance of the matrix material is detected by an atomic force microscope in a tapping mode, and the surface of the fusion protein modified glass sheet is a compact and compact protein modification layer and is regular and ordered.

Example 4: fusion protein adhesion test

The adhesion mechanism of mussel byssus protein is always the popular research field, the strong adhesion force is considered to be closely related to the amino acid residues, the most important is DOPA residue modified by hydroxylation of tyrosine, DOPA can form strong hydrogen bonds with polar polymers such as protein, and the phenol group of DOPA has strong metal complexing force and can form irreversible organic metal complex on the surface of the material. Studies have shown that adhesion is significantly reduced after removal of the DOPA residues at the adhesion interface. Therefore, the presence of DOPA largely determines the adhesion. The presence or absence of the DOPA residue can be detected by NBT, and the mussel byssus protein containing DOPA becomes blue obviously when meeting NBT. We compare the diblock fusion protein of this study with non-hydroxylated mussel byssus protein, and add NBT separately, the results show that the diblock fusion protein contains DOPA and is adhesive. As shown in FIG. 4, the left side is the non-hydroxylated mussel byssus protein, and the right side is the diblock fusion protein, so that it is clear that the color of the right side is dark.

Example 5: fusion protein freezing resistance test

Culturing the recombinant strain in LB culture medium at 37 deg.C and 220r/min to OD₆₀₀The resulting suspension was centrifuged (5000r/rain, 4min) at 0.6. Divided into three groups, and the diblock fusion proteins MP-AFP, BSA and LB are respectively added for heavy suspension precipitation. The cells were incubated at-20 ℃ for 0, 24, 48, 72, 96, 120 and 144h, 100uL of the suspension was applied to LB plates, 5 replicates of each treatment were performed, and the number of colonies was counted after overnight incubation at 37 ℃. The antifreeze efficiency was calculated by taking the number of colonies at 0h as 100%. The results are shown in FIG. 5. It can be obviously seen that the survival rate of the bacteria preserved by using the diblock fusion protein (MP-AFP) is far higher than that of the bacteria preserved by using BSA and LB, which shows that the fusion protein has good freezing resistance.

Example 6: MTT cytotoxicity assay

(ii) animal cell culture

The MTT cytotoxicity detection is carried out by taking NIH/3T3 fibroblasts as experimental animal cells. DMEM medium containing 1% penicillin and streptomycin and 10% calf serum at 37 deg.C and 5% CO₂NIH/3T3 fibroblasts were cultured under the conditions.

② aseptic treatment of the matrix Material

The modified mica plates were washed 3 times with sterile PBS solution for aseptic processing.

(iii) MTT test

3.5 x 10^4 cells per well were plated in 48-well plates and cultured for 12 h. The sterilized mica plate is added into the hole correspondingly, and the NIH/3T3 cells are cultured for 24 h. After the completion of the incubation, 300. mu.L of 10% MTT solution was added to each well, and the incubation was continued for 4 hours. After the incubation, the supernatant was aspirated from the well plate, 330. mu.L of DMSO solution was added to each well, and shaken on a shaker for 10 min. The above liquid was transferred to a 96-well plate, and absorbance was measured at 490 nm. The cell wells incubated with untreated mica plates were used as blank control and set as 100%, and it was found that the mica plates modified with the fusion protein did not have any toxic effect on the cells, and the survival rate was higher than 100%.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing examples, or some technical features may be equivalently replaced, to finally prepare a fusion protein. It should be understood that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Nucleotide sequence table:

SEQ ID NO.1：

AKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKSSEEYKGGYYPGNTYHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKKYYGGGSSGGGGSAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYK

SEQ ID NO.2：

MGFKTCGFSKKWLVTAVIVMCLCTECYCQCTGGADCTSCTAACTGCGNCPNAVTCTNSQHCVKATTCTGSTDCNTAVTCTNSKDCFEAQTCTDSTNCYKATACTNSTGCPGH*

SEQ ID NO.3：

GGATCCMAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKSSEEYKGGYYPGNTYHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKKYYGGGSSAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKGGGGSGGGGSGGGGSMGFKTCGFSKKWLVTAVIVMCLCTECYCQCTGGADCTSCTAACTGCGNCPNAVTCTNSQHCVKATTCTGSTDCNTAVTCTNSKDCFEAQTCTDSTNCYKATACTNSTGCPGH*GCGGCCGC

SEQ ID NO.4：

gctaaaccgt cttacccgcc gacctacaaa gctaaaccgt cttacccgcc gacctacaaa gctaaaccgt cttacccgcc gacctacaaa gctaaaccgt cttacccgcc gacctacaaa gctaaaccgt cttacccgcc gacctacaaa gctaaaccgt cttacccgcc gacctacaaa tcttctgaaga atacaaaggt ggttactacc cgggtaacac ctaccactac cactctggtg gttcttaccac ggttctggtta ccacggtggt tacaaaggta aatactacgg taaagctaaa aaatactact acaaatacaa aaactctggt aaatacaaat acctgaaaaa agctcgtaaa taccaccgta aaggttacaa aaaatactac ggtggtggtt cttctgctaaa ccgtcttacc cgccgacct acaaagctaa accgtcttac ccgccgacc tacaaagct aaaccgtctt acccgccga cctacaaag ctaaaccgt cttacccgcc gacctacaa agctaaacc gtcttaccc gccgaccta caaagctaa accgtcttac ccgccgac ctacaaagg tggtggtgg ttctggtggt ggtggttctg gtggtggtg gttctatggg attcaaaacg tgtggtttttc aaaaaaatg gttagtaaca gcagttatag ttatgtgtttgt gtaccgagt gttattgcca atgcactgga ggtgctgatt gtactagttg tacagcagca tgcactggtt gtggaaactg tccaaatgca gtaacgtgta ccaattctca acattgtgtc aaggcaacaa catgtactgg gtctacagat tgtaatacag ccgtgacgtg tacaaactca aaagactgtt tcgaagccca aacatgtact gactcaacca actgttacaa agctacagcc tgtaccaatt caacaggatg tcccggacat taa

sequence listing

<110> Tianjin university

<120> diblock fusion protein with adhesion-anti-freezing dual functions, and synthesis method and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 201

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro

1 5 10 15

Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys

20 25 30

Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr

35 40 45

Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu Glu

50 55 60

Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly

65 70 75 80

Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr

85 90 95

Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys

100 105 110

Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys

115 120 125

Lys Tyr Tyr Gly Gly Gly Ser Ser Gly Gly Gly Gly Ser Ala Lys Pro

130 135 140

Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr

145 150 155 160

Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr

165 170 175

Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala

180 185 190

Lys Pro Ser Tyr Pro Pro Thr Tyr Lys

195 200

<210> 2

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Gly Phe Lys Thr Cys Gly Phe Ser Lys Lys Trp Leu Val Thr Ala

1 5 10 15

Val Ile Val Met Cys Leu Cys Thr Glu Cys Tyr Cys Gln Cys Thr Gly

20 25 30

Gly Ala Asp Cys Thr Ser Cys Thr Ala Ala Cys Thr Gly Cys Gly Asn

35 40 45

Cys Pro Asn Ala Val Thr Cys Thr Asn Ser Gln His Cys Val Lys Ala

50 55 60

Thr Thr Cys Thr Gly Ser Thr Asp Cys Asn Thr Ala Val Thr Cys Thr

65 70 75 80

Asn Ser Lys Asp Cys Phe Glu Ala Gln Thr Cys Thr Asp Ser Thr Asn

85 90 95

Cys Tyr Lys Ala Thr Ala Cys Thr Asn Ser Thr Gly Cys Pro Gly His

100 105 110

<210> 4

<211> 338

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gly Gly Ala Thr Cys Cys Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr

1 5 10 15

Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr

20 25 30

Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala

35 40 45

Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro

50 55 60

Thr Tyr Lys Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn

65 70 75 80

Thr Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His

85 90 95

Gly Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr

100 105 110

Lys Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys

115 120 125

Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Ala

130 135 140

Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro

145 150 155 160

Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro

165 170 175

Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr

180 185 190

Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Gly Gly Gly Gly Ser

195 200 205

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Gly Phe Lys Thr Cys

210 215 220

Gly Phe Ser Lys Lys Trp Leu Val Thr Ala Val Ile Val Met Cys Leu

225 230 235 240

Cys Thr Glu Cys Tyr Cys Gln Cys Thr Gly Gly Ala Asp Cys Thr Ser

245 250 255

Cys Thr Ala Ala Cys Thr Gly Cys Gly Asn Cys Pro Asn Ala Val Thr

260 265 270

Cys Thr Asn Ser Gln His Cys Val Lys Ala Thr Thr Cys Thr Gly Ser

275 280 285

Thr Asp Cys Asn Thr Ala Val Thr Cys Thr Asn Ser Lys Asp Cys Phe

290 295 300

Glu Ala Gln Thr Cys Thr Asp Ser Thr Asn Cys Tyr Lys Ala Thr Ala

305 310 315 320

Cys Thr Asn Ser Thr Gly Cys Pro Gly His Gly Cys Gly Gly Cys Cys

325 330 335

Gly Cys

<210> 4

<211> 972

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gctaaaccgt cttacccgcc gacctacaaa gctaaaccgt cttacccgcc gacctacaaa 60

gctaaaccgt cttacccgcc gacctacaaa gctaaaccgt cttacccgcc gacctacaaa 120

gctaaaccgt cttacccgcc gacctacaaa gctaaaccgt cttacccgcc gacctacaaa 180

tcttctgaag aatacaaagg tggttactac ccgggtaaca cctaccacta ccactctggt 240

ggttcttacc acggttctgg ttaccacggt ggttacaaag gtaaatacta cggtaaagct 300

aaaaaatact actacaaata caaaaactct ggtaaataca aatacctgaa aaaagctcgt 360

aaataccacc gtaaaggtta caaaaaatac tacggtggtg gttcttctgc taaaccgtct 420

tacccgccga cctacaaagc taaaccgtct tacccgccga cctacaaagc taaaccgtct 480

tacccgccga cctacaaagc taaaccgtct tacccgccga cctacaaagc taaaccgtct 540

tacccgccga cctacaaagc taaaccgtct tacccgccga cctacaaagg tggtggtggt 600

tctggtggtg gtggttctgg tggtggtggt tctatgggat tcaaaacgtg tggtttttca 660

aaaaaatggt tagtaacagc agttatagtt atgtgtttgt gtaccgagtg ttattgccaa 720

tgcactggag gtgctgattg tactagttgt acagcagcat gcactggttg tggaaactgt 780

ccaaatgcag taacgtgtac caattctcaa cattgtgtca aggcaacaac atgtactggg 840

tctacagatt gtaatacagc cgtgacgtgt acaaactcaa aagactgttt cgaagcccaa 900

acatgtactg actcaaccaa ctgttacaaa gctacagcct gtaccaattc aacaggatgt 960

cccggacatt aa 972

Claims

1. A diblock fusion protein with adhesion-freeze resistance dual functions is characterized by containing mussel adhesion protein and freeze resistance protein; the amino acid sequence of the fusion protein is SEQ ID NO. 3; wherein the tyrosine residue in the fusion protein is partially converted into the unnatural amino acid DOPA.

2. The fusion protein of claim 1, wherein the mussel adhesion protein has the sequence shown in SEQ ID No. 1.

3. Fusion protein according to claim 1, characterized in that the antifreeze protein has the sequence shown in SEQ ID No. 2.

4. The fusion protein of claim 1, characterized in that the nucleotide sequence is SEQ ID No. 4.

5. The method for synthesizing the diblock fusion protein having the adhesion-anti-freeze dual function according to claim 1, wherein: cloning the gene of the fusion protein to a plasmid vector to obtain a recombinant expression plasmid, transforming the recombinant expression plasmid into a host cell, inducing the host cell to excessively express the fusion protein by using an inducer, and separating and purifying to obtain the fusion protein.

6. The method of synthesizing a fusion protein according to claim 5, comprising the steps of:

(2) cloning the synthesized diblock fusion protein gene into an escherichia coli expression vector pET-28a, adding a BamHI restriction site at the 5 'end of the fusion protein gene, and adding a Not I restriction site at the 3' end of the gene to obtain a recombinant expression vector;

(3) constructing an expression vector for expressing tyrosinase;

(4) the successfully constructed recombinant expression vector and the expression vector for expressing tyrosinase are jointly transformed into escherichia coli BL21(DE3), and an LB solid culture medium containing kanamycin and chloramphenicol dual resistance is utilized; screening transformants by using a plate;

(6) and (3) separating and purifying the diblock fusion protein.

7. The method of synthesizing a fusion protein according to claim 6, wherein the host cell expressing the fusion protein is a host cell comprising the nucleotide sequence of SEQ ID No.4 or a host cell comprising a recombinant expression vector.

8. The method for synthesizing the fusion protein according to claim 6, wherein the host cell is Escherichia coli BL21(DE 3); the recombinant expression vector is obtained by cloning the fusion protein gene into an escherichia coli expression vector pET-28a, and contains BamH I and Not I restriction enzyme sites, a T7 strong promoter, a lac lactose operon, a hexahistidine tag and resistance screening genes of kanamycin and chloramphenicol.