CN104628845B - A kind of Cyanea capillata serpin and its encoding gene and application - Google Patents

Abstract

The invention belongs to biomedicine technical field, has not yet to see the relevant research report to jellyfish Kazal type serpins.The invention provides a kind of Cyanea capillata Kazal type serpins and preparation method thereof, described Cyanea capillata serpin, there is such as SEQ ID NO:The protein of amino acid sequence composition shown in 2.Present invention also offers the encoding gene of Cyanea capillata serpin, has such as SEQ ID NO:Nucleotide sequence shown in 1.Meanwhile the application present invention also offers Cyanea capillata serpin and its encoding gene in antibacterials, anti-inflammation drugs, shielding medicine for skin etc. is prepared.

Description

A kind of jellyfish jellyfish serine protease inhibitor and its coding gene and application

technical field

The invention belongs to the technical field of biomedicine, and specifically relates to a jellyfish serine protease inhibitor and its coding gene and application.

Background technique

The ocean is a treasure house of biological resources, and more than 80% of the species in the world are stored in the ocean. Due to long-term living in the special marine ecological environment of high salinity, high pressure and anoxic environment, the physiological structure and metabolites of marine organisms are different from those of land organisms, and they produce and accumulate a large number of substances with special chemical structures, special physiological activities and functions. An important resource for the development of new marine medicines. Marine biological resources mainly include marine biotoxins, physiologically active substances, biological information substances and biological functional materials. Marine bioactive substances are an important part of marine biological resources. So far, more than 3,000 new types of active substances have been discovered in marine organisms, many of which have been proven to have anti-tumor, anti-virus, antibacterial, and anti-cardiovascular properties. Vascular disease, anti-fatigue, and immune-boosting effects. The development and utilization of marine bioactive substances has become a hot spot in the research of modern medicine and health food. At present, the research on the activity of marine biological resources mainly focuses on antitumor and antibacterial aspects, while marine biological resources with significant biological activities mainly include marine microorganisms and marine invertebrates (such as sea squirts, sponges, jellyfish, sea anemones, sea hares, etc. ).

There are many kinds of marine microorganisms, according to statistics, there are 2 million to 200 million species. Marine invertebrates lack the diverse antibodies of the acquired immune system and can only rely on the innate immune system to resist invading bacteria or pathogens. Under the long-term natural selection pressure and complex marine environment, the innate immune system of marine invertebrates is very effective and powerful. Studies have found that marine invertebrates are very rich in endogenous antibacterial active substances, and have become an important resource for the development of new antibacterial drugs. Jellyfish belong to the phylum Cnidaria, which is a kind of marine plankton with a wide distribution and a very large biomass. Jellyfish are rich in a variety of biologically active substances, including jellyfish toxin protein and some new functional proteins with strong activity and good development prospects. Previous studies have found that jellyfish also contain more antimicrobial peptides and lectins, which is of great significance for jellyfish living in complex seawater surface environments to resist the invasion of external bacteria and viruses. It is suggested that active substances with antiviral and antibacterial effects may be obtained from jellyfish.

Serine protease inhibitor (serine protease inhibitor) is a general term for a class of polypeptide serine protease inhibitors with a common source (more than 500 family members), widely distributed in eukaryotes and viruses, by regulating the activity of serine proteases, participating in Many important life activities such as fibrinolysis, inflammatory response, cell migration, cell differentiation and cell apoptosis. In addition, proteases that function in the coagulation and complement systems are also regulated by serine protease inhibitors to prevent their protease activity from causing damage to surrounding tissues. In recent years, studies have found that serine protease inhibitors also play an important role in the process of innate immune defense. It is known that most pathogenic microorganisms of animals and plants will secrete proteases into the host body, destroying the host's physical defense to further strengthen the infection. Serine protease inhibitors in the host participate in the host's natural immune defense process by inhibiting the protease activity secreted by pathogenic microorganisms.

Serine protease inhibitors mainly include: α-macroglobulin, serpin, Kunitz and Kazal and other types.

At present, some studies on Kazal-type inhibitors in aquatic animals have found that Kazal-type serine protease inhibitors have obvious antibacterial activity. For example, SPIPm2, an inhibitor containing 5 Kazal domains found in the blood cells of Penaeus monodon, has a significant inhibitory activity on subtilisin, and it is speculated that it may play an immune role in the infection process of pathogenic bacteria (see literature: Somprasong N, Rimphanitehay A, Kittassanakajon A.A five-domain Kazal-type Serine proteinase inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities[J].Developmental and Comparative Immunology,2006,30(11):998-1008.); in China The inhibitor FcSPI-1 containing 9 Kazal domains was found in the hepatopancreas of prawn (Fenneropenaeus chinensis), and studies have shown that it not only plays a role in the infection of pathogenic microorganisms, but also participates in the regulation of its own protease activity (see literature: Wang Z H, Zhao X F, Wang J X.Characterization, kinetics, and possible function of Kazal-type proteinase inhibitors of Chinese white shrimp, Fenneropenaeuschinensis[J].Fish&Shellfish Immunology,2009,26(6):885-897.); in Hydra The inhibitor Kazal2 with 3 Kazal domains found in has a strong ability to kill Staphylococcus aureus (see literature: Augustin R, Siebert S, Boscht C. Identification of a Kazal-type serine protease inhibitor with potent anti- staphylococcal activity as part of Hydra's innateimmune system[J].Developmental and Comparative Immunology,2009,33(7):830-837.); rFc-Kazal found in Chinese prawn (Fenneropenaeus chinensis) Staphylococcus aureus, Aeromonas salmonicida, Bacillus thuringiensis All have a certain antibacterial effect (see literature: Huang Ming, Liu Yichen, Zhang Yichen, etc. Recombinant expression and activity analysis of the Kazal-type serine protease inhibitor gene (Fc-Kaza1) of Penaeus prawn in China[J].Acta Fisheries, 2011, 35(9):1310-1318.).

At present, there are no relevant research reports on jellyfish Kazal-type serine protease inhibitors at home and abroad.

The inventor's research group has been working on the extraction and research of active components in jellyfish, and has obtained a Chinese invention patent: patent number ZL201310189933.5, and the name of the invention is "a type of thioredoxin from jellyfish and its coding gene and application", authorized announcement number CN103232979B; patent number ZL201310188702.2, the title of the invention is "a jellyfish peroxide reductase and its coding gene and application", authorized announcement number CN103255113B; Chinese patent has been applied: application number 201310258184.7, the title of the invention is "A jellyfish astaxanthin-like metalloproteinase CALP1 and its coding gene and expression method", application publication number CN 104195124A; application number 201310629343.X, the title of the invention is "a jellyfish cardiovascular toxin Preparation method of crude extract", application publication number: CN103613653A; application number 201310628068.X, the title of the invention is "a preparation method of jellyfish hemolytic toxin crude extract", application publication number CN103626861A, etc.

Contents of the invention

The purpose of the present invention is to provide a jellyfish serine protease inhibitor and its coding gene, another purpose of the present invention is to provide the preparation method of the jellyfish serine protease inhibitor, the third purpose of the present invention is to provide the Application of a jellyfish serine protease inhibitor in the preparation of antibacterial drugs, anti-inflammatory drugs, skin protection agents and the like.

The invention obtains the coding gene of the jellyfish serine protease inhibitor by constructing a jellyfish jellyfish tentacle tissue cDNA library, and measuring and annotating the sequence of the recombinant clone of the library. The jellyfish serine protease inhibitor is the first jellyfish serine protease inhibitor with obvious antibacterial activity discovered, and it is an important active component of the innate immune system in jellyfish. The protein has good application in the research of antibacterial and anti-inflammatory drugs prospect.

The main technical scheme of the present invention is, by constructing the cDNA library of jellyfish tentacle tissue, sequencing and screening it, obtaining the coding gene of jellyfish serine protease inhibitor and carrying out recombinant expression, and then inhibiting serine proteases activity and antibacterial activity were studied.

The first aspect of the present invention provides a jellyfish serine protease inhibitor, said a jellyfish serine protease inhibitor, has the following (i) or (ii) protein:

(i) a protein composed of the amino acid sequence shown in SEQ ID NO:2;

(ii) A protein derived from (i) in which the amino acid sequence shown in SEQ ID NO: 2 is substituted, deleted and/or added by one or several amino acids and has equivalent functions.

Described a jellyfish serine protease inhibitor, such as the amino acid sequence shown in SEQ ID NO: 2, the protein contains a signal peptide, is a secreted protein, is located outside the cell, the molecular weight is 19.02kDa, and the isoelectric point is 8.75.

The present invention also provides a kind of coding gene of jellyfish serine protease inhibitor, which is the DNA molecule of following (i) or (ii):

(i) the nucleotide sequence shown in SEQ ID NO:1;

(ii) A nucleotide sequence having more than 80% homology with the nucleotide sequence shown in SEQ ID NO:1.

The full length of the nucleotide sequence of the said serine protease inhibitor gene of Aequida habilis is 773bp, as shown in SEQ ID NO:1.

The product of the invention is a Kazal-type serine protease inhibitor, which contains three typical Kazal domains, each of which includes 50-60 amino acid residues and six cysteines forming three pairs of disulfide bonds. The inhibitory specificity of Kazal inhibitors is determined by the second amino acid P1 after the second cysteine in the Kazal domain, so domains containing different P1 amino acid residues have different inhibitory specificities. P1 of the serine protease inhibitor in the present invention is alanine and arginine, which are presumed to have the activity of inhibiting elastase and trypsin.

A second aspect of the present invention provides a method for preparing a jellyfish serine protease inhibitor, the method comprising the steps of:

(A) Construction of a jellyfish tentacle cDNA library;

(B) Determining and analyzing the sequence of the cDNA library recombinant clone of the above-mentioned jellyfish tentacle tissue cDNA library to obtain a nucleotide sequence encoding a serine protease inhibitor;

(C) Constructing the expression plasmid of A. phagina serine protease inhibitor, recombinant engineering bacterium;

(D) Expression of a jellyfish serine protease inhibitor;

(E) Purification of recombinant Aequoria habilis serine protease inhibitors.

The jellyfish tentacle tissue cDNA library in the step (A) is constructed by the following method:

1) extracting total RNA from the tentacle tissue of Jellyfish jellyfish;

2) Isolating mRNA and synthesizing the cDNA of the tentacle tissue of Jellyfish jellyfish;

3) The above cDNA was inserted into the pUC19 plasmid vector, and then transformed into Escherichia coli DH5α, spread on the LB medium plate for blue-white screening, and then the cDNA library of the tentacle tissue of A. jellyfish was constructed.

In the step (C), the expression plasmid of constructing the Aequosia habilis serine protease inhibitor, the recombinant engineering bacterium, the specific steps are:

1) Design a pair of PCR primers with specific restriction sites EcoRI and XhoI according to the sequence of the serine protease inhibitor gene of A. phagina japonicus and the restriction site of the prokaryotic expression vector pGEX-6P-1, as follows:

Upstream primer: 5'CGGAATTCATGACCAAGCCATT 3' (SEQ ID NO:3)

Downstream primer: 5'CCGCTCGAGTTTTCTGCATTG 3'(SEQ ID NO:4)

The PCR reaction conditions are: 95°C for 30 sec; 95°C for 30 sec, 55.5°C for 30 sec, 68°C for 1 min, 38 cycles; 68°C for 5 min;

2) Recovered PCR product and pGEX-6P-1 prokaryotic expression plasmid;

3) The coding sequence was connected to the pGEX-6P-1 vector by using two restriction sites to construct a recombinant expression plasmid, and then transformed into Escherichia coli Rosetta (DE3).pLysS to obtain recombinant engineering bacteria.

The expression condition of the Aequorea habilis serine protease inhibitor in the step (D) is: induction for 7 hours at 12° C., 1 mMIPTG, and 150 rpm, under which the recombinant protein can be expressed in a soluble form.

The purification in the step (E) is obtained by one-step purification using a GSTrap HP 4B FF affinity chromatography column, and the elution conditions are 90% of the total volume of the binding buffer and 10% of the elution buffer; wherein, The binding buffer was 1.8 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, pH 7.3, and the elution buffer was 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0.

The preparation method of the invention is simple and low in cost.

The third aspect of the present invention provides the application of the jellyfish serine protease inhibitor and its coding gene in the preparation of antibacterial drugs, anti-inflammatory drugs and skin protection agents.

The present invention further provides the application of said jellyfish serine protease inhibitor and its coding gene in the preparation of subtilisin A inhibitor, proteinase K inhibitor, elastase inhibitor and the like.

The invention provides the application of the jellyfish serine protease inhibitor and its coded gene in the preparation of antibacterial drugs, and the bacteria are Staphylococcus aureus, Bacillus subtilis, Candida albicans, Escherichia coli, Vibrio vulnificus and the like.

The present invention provides the application of the jellyfish jellyfish serine protease inhibitor and its coded gene in the preparation of anti-inflammatory drugs. Escherichia coli and Vibrio vulnificus etc. grow, so Aequola habilis serine protease inhibitor can be used for preparing anti-inflammatory drug, and described inflammation is the infection that pathogenic bacteria Staphylococcus aureus, Vibrio vulnificus and conditional pathogenic bacteria Escherichia coli cause Wait.

The invention provides the application of the jellyfish jellyfish serine protease inhibitor and its coded gene in the preparation of a skin protectant. The skin protectant is used to prevent infection after stings by marine organisms and the like. On the one hand, the jellyfish serine protease inhibitor of the present invention can utilize the inhibitory effect of the protease inhibitor on protease to weaken the damage of pathogenic microorganisms to the human body; at the same time, because the serine protease protease inhibitor also has bacteriostasis, it can prevent microbial infection . The type of skin protectant can be a component of a skin protectant for jellyfish stings, or a component of a skin protectant for preventing marine organism wounds and open wounds from being infected by marine microorganisms after seawater immersion.

The Kazal type serine protease inhibitor of the recombinant jellyfish jellyfish obtained by the invention has remarkable serine protease inhibitory activity and antibacterial activity. In the experiment of detecting the inhibitory ability of the recombinant protein to serine proteases, with the increase of the concentration of the recombinant protein of Aequorea habilis serine protease inhibitors, the inhibition rate of serine proteases in the reaction system increased significantly. In the experiment of detecting the combination of recombinant serine protease inhibitors from jellyfish and microorganisms, it was found that Kazal-type serine protease inhibitors had weak binding activity to Staphylococcus aureus, Bacillus subtilis, Candida albicans, Escherichia coli and Vibrio parahaemolyticus, It has strong binding activity with Candida albicans and Vibrio vulnificus. In the experiment of detecting the inhibitory ability of recombinant protein to microorganisms, with the increase of the concentration of recombinant protein serine protease inhibitor protein from Aequoria phaformis, its effect on Staphylococcus aureus, Bacillus subtilis, Candida albicans, Escherichia coli, Vibrio vulnificus, etc. The growth inhibition effect of microorganisms was significantly enhanced. This experiment confirmed the inhibitory ability and antibacterial activity of the recombinant Kazal-type serine protease inhibitors of jellyfish phaformis on serine proteases.

Therefore, the present invention has recombined the Kazal-type serine protease inhibitor of A. jellyfish for the first time and found that it has significant antibacterial activity. In the present invention, the Kazal-type serine protease inhibitor derived from A. jellyfish has significant development value. Anti-inflammatory drugs, cosmetics, food and agriculture industries have certain application potential. The jellyfish serine protease inhibitor involved in the present invention will have great application value in the development of antibacterial drugs, anti-inflammatory drugs and skin protective agents.

Description of drawings

Figure 1 is a multiple alignment of the sequences of Cyanea capillata Kazal-type serine protease inhibitors and other species of Kazal-type serine protease inhibitors. Among them, black represents completely homologous regions.

Fig. 2 is the electrophoresis result of the gradient PCR product of the coding sequence of the open reading frame of the jellyfish jellyfish serine protease inhibitor in the present invention. Among them, M: nucleic acid molecular weight marker; 1-12: PCR product.

Fig. 3 is the SDS-PAGE electrophoresis result of the expression induced by the recombinant jellyfish serine protease inhibitor in the present invention. Among them, M: protein molecular weight marker; 1: uninduced recombinant Escherichia coli; 2: ultrasonic lysate after induction with 1mM IPTG at 16°C for 7 hours; 3: ultrasonic supernatant after induction with 1mM IPTG at 16°C for 7 hours; 4: ultrasonic supernatant after induction with 1mM IPTG 16 Ultrasonic precipitation after induction at ℃ for 7 hours; 5: Ultrasonic lysate after induction with 1mM IPTG at 20℃ for 7 hours; 6: Ultrasonic supernatant after induction with 1mM IPTG at 20℃ for 7 hours; 7: Ultrasonic precipitation after induction with 1mM IPTG at 20℃ for 7 hours; red box Indicates the location of the recombinant protein.

Fig. 4 is the SDS-PAGE electrophoresis result of the separation and purification of the recombinant jellyfish serine protease inhibitor in the present invention. Among them, 1: recombinant E. coli before induction; 2: ultrasonic lysate of recombinant E. coli after induction 3: supernatant of ultrasonic lysis of recombinant E. coli after induction; 4: breakthrough peak during purification of recombinant protein; 5: purification of recombinant protein Peak eluting at time; red box indicates position of recombinant protein.

Fig. 5 is the result of detecting the inhibitory ability of the recombinant jellyfish serine protease inhibitor to serine proteases. Wherein A is the inhibitory ability to subtilisin A, B is the inhibitory ability to proteinase K, and C is the inhibitory ability to elastase. The results showed that the molar ratios of the serine protease inhibitors from Aequola habilis to the three serine proteases when they reached the maximum inhibitory activity were: 2000:1 (subtilisin A), 2000:1 (proteinase K) and 80:1 ( elastase). The inhibitory effect of the serine protease inhibitor from Aequoria phagoides was enhanced with the increase of its concentration, and its inhibitory effect did not increase after reaching a certain concentration, while no inhibitory activity was detected for trypsin and chymotrypsin (results not shown).

Fig. 6 is the detection result of the binding experiment of the recombinant jellyfish serine protease inhibitor and bacteria. The upper row shows the protein eluted by SDS, and the lower row shows the bacterial protein after TBS elution. Nos. 1-9 are the uninduced group (negative control group), the purified serine protease inhibitor of Aequorea habilis (positive control group), Staphylococcus aureus, Bacillus subtilis, Candida albicans, Escherichia coli, parahaemolyticus Vibrio, Vibrio alginolyticus and Vibrio vulnificus. The results showed that Kazal-type serine protease inhibitors had strong binding activity to Candida albicans and Vibrio vulnificus, weak binding activity to Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Vibrio parahaemolyticus, and no binding activity to Vibrio alginolyticus. Binding activity detected.

Fig. 7 is the result of detecting the inhibitory ability of the recombinant Aequoria habilis serine protease inhibitor to the growth of microorganisms. It can be seen that the serine protease inhibitors of A. aeruginosa have obvious growth inhibition on Staphylococcus aureus (A), Bacillus subtilis (B), Candida albicans (C), Escherichia coli (D) and Vibrio vulnificus (E) active.

detailed description

The present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings. However, the following examples should not be considered as limiting the scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The Cyanea capillata selected in the present invention was collected from Sanmen Bay, Zhejiang Province, and was identified by Professor Hong Huixin from the Fisheries College of Jimei University (L. Xiao et al, Toxicon, 2009, 53:146-152).

Example 1: Screening and sequence analysis of jellyfish serine protease inhibitors

1) The extraction of total RNA from the tentacles of A. phagina jellyfish was carried out according to the instructions of the Trizol kit from Invitrogen, and the protein was removed with chloroform to obtain about 7 μg of total RNA;

2) The isolation of mRNA was carried out according to the instructions of Oligotex mRNA Spin-column Kit of QIANGEN Company, and the synthesis of cDNA was carried out according to the instructions of SMART cDNA Library Construction Kit of Clontech Company;

3) Insert the cDNA into the pUC19 plasmid vector (purchased from Takara Company), and then transform it into Escherichia coli DH5α (purchased from Beijing Bomaide Company), spread it on a 15cm petri dish for blue-white screening, and then form the tentacles of jellyfish jellyfish Tissue cDNA library.

The library has a total of 1923 colonies, including 35 coeruleus, a recombination rate of 98.18%, a library capacity of 1.92*106, and 1035 effective sequences with an insertion length of ≥ 400bp. These sequences were merged into unigene using the principle of 100bp and 90% to obtain unigene The number is 528. At the same time, the full-length analysis of the sequences shows that 12 of the 20 sequences have relevant homology information, and the result is that 12 of them are full-length, and the integrity ratio: 12/12×100%=100%, indicating that the cDNA library has better quality. The cDNA library was randomly sequenced, and the resulting sequence was subjected to BLASTx analysis (http://blast.ncbi.nlm.nig.gov) after removing the vector.

4) The serine protease inhibitor gene of Aequorea habilis comes from the clone numbered 1E09 in the above cDNA library. The full-length sequence is 773bp, including an open reading frame of 531bp, encoding a protein with 176 amino acid residues, a molecular weight of 19.02kDa, and an isoelectric point of 8.75. Blastx search results show that the protein is highly homologous to Kazal-type serine protease inhibitors of multiple species (as shown in Figure 1), and is a new molecule of the Kazal-type serine protease inhibitor family derived from jellyfish. Further bioinformatics analysis showed that the protein contained a signal peptide, was a secreted protein, and was located outside the cell.

Example 2: Construction of a recombinant expression plasmid for Aequosia habilis serine protease inhibitors and recombination of engineering bacteria

1) A pair of primers were designed and synthesized according to the sequence of the serine protease inhibitor gene sequence of A. jellia habilis and the restriction site of the prokaryotic expression vector pGEX-6P-1 (purchased from Novagen), wherein the upstream primer contains the restriction site EcoR I (GAATTC), the downstream primer contains restriction site XhoI (CTCGAG), the sequences of the two primers are as follows:

Upstream primer: 5'CGGAATTCATGACCAAGCCATT 3' (SEQ ID NO:3)

Downstream primer: 5'CCGCTCGAGTTTTCTGCATTG 3'(SEQ ID NO:4)

After the gradient PCR experiment, 55.5°C was selected as the optimal annealing temperature, and the target gene was amplified by PCR. The PCR reaction conditions were: 95°C for 5 min; 95°C for 30 sec, 55.5°C for 30 sec, 68°C for 1 min, 38 cycles; keep at 68°C for 5 minutes. After the PCR reaction, PCR products containing NdeI and XhoI restriction sites at the 5' end were obtained, and nucleic acid electrophoresis showed that the PCR product band was in the correct position (as shown in Figure 2), about 528 bp, and the PCR product was recovered;

2) Digest the recovered PCR product and pGEX-6P-1 prokaryotic expression plasmid with NdeI and XhoI endonucleases from NEB Company according to the product instructions;

3) After recovering the digested product, carry out the ligation reaction according to the T4DNA ligase instructions of NEB Company, transform the ligation reaction product into Escherichia coli Rosetta (DE3). Incubate for 16 hours on a Petri dish containing chloramphenicol (100 μg/ml) and chloramphenicol (34 μg/ml). Pick a single colony and culture it in 5ml of LB medium containing ampicillin (100μg/ml) and chloramphenicol (34μg/ml) for 12 hours. After the successful recombination is identified by bacterial liquid PCR and plasmid digestion verification, etc. , using pGEX F/R as a sequencing primer to carry out two-way sequencing on the recombinant plasmid, and verify that the cloned gene is the target gene, indicating that the recombinant expression plasmid of Aequoria habilis serine protease inhibitor was correctly constructed.

Example 3: Expression of Serine Protease Inhibitors from Aequorea habilis

Add the recombinant Escherichia coli Rosetta (DE3).pLysS bacterial liquid with correct sequencing to the liquid LB medium containing ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml), and cultivate it to OD at 37°C and 250 rpm on a shaker When the ₆₀₀ was 0.6-0.8, the inducer IPTG was added for induction.

The optimal expression condition of the recombinant protein was determined as follows: induction at 12° C., 1 mM IPTG, and 150 rpm for 7 hours. After induction, the bacteria were collected by centrifugation, and SDS-PAGE electrophoresis showed that the recombinant protein could be expressed in a soluble form under this induction condition, and the molecular weight was consistent with the predicted value (about 45kDa after adding the GST tag), as shown in Figure 3.

Embodiment 4: Purification of recombinant jellyfish serine protease inhibitor

Centrifuge the induced bacterial liquid (10000×g*10min) to collect the bacterial cells, and then use the binding buffer (1.8mMKH2PO4, 140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, pH 7.3) to fully resuspend the bacterial cells for ultrasonic lysing , and the supernatant was obtained after ultrasonic lysate centrifugation (10000×g*10min) for purification. Finally, according to GE Healthcare's GSTTrap HP 4B FF chromatography column and Operation instructions of the separation and purification system Perform affinity chromatography to obtain a relatively pure serine protease inhibitor from Aequorea habilis.

SDS-PAGE electrophoresis (as shown in Figure 4) shows that the target band matches the predicted position, and the purity is more than 90%. The elution conditions used are: 90% binding buffer and 10% elution buffer (50mM Tris-HCl , 10 mM reduced glutathione, pH 8.0).

Example 5: Detection of Serine Protease Activity Inhibited by Recombinant Aequorea habilis Serine Protease Inhibitor

After the relatively single target protein was obtained by the affinity chromatography method in Example 4, the proteases used in the protease activity inhibition experiment were: subtilisin A, proteinase K, chymotrypsin, trypsin and elastase. All reagents and substrates in this experiment were prepared in binding buffer. Experimental steps:

Step 1: Add 50 μl inhibitor and 150 μl protease (100 mM Tris-HCl pH 8.0) to a 96-well plate, incubate at 25°C for 10 min, and measure the absorbance value OD1 at 405 nm;

Step 2: Add 10 μl chromogenic substrate and incubate for 5 minutes;

The third step: stop the reaction with 50 μl 50% (v/v) acetic acid, and measure the absorbance value OD2 at 405 nm;

Actual absorbance value = OD2-OD1;

Result: average of three results

Ri=1-(OD405 with inhibitor-OD405 control)/(OD405 without inhibitor-OD405 control) The reaction system is shown in Table 1:

The obtained data is used as a dose-response curve, the ordinate is the remaining reactivity of the protease, and the abscissa is the molar ratio of the serine protease inhibitor to the protease in the reaction system.

The experimental results are shown in Figure 5. As the concentration of the recombinant serine protease inhibitor increases, the remaining enzyme activity in the reaction system decreases, and the inhibition rate of the inhibitor on subtilisin, proteinase K and elastase increases significantly.

Example 6: Detection of the ability of recombinant jellyfish serine protease inhibitors to bind microorganisms

After the relatively single target protein was obtained by the affinity chromatography method in Example 4, the binding ability of the protein to microorganisms was detected. The microorganisms used in the experiment are: Staphylococcus aureus, Bacillus subtilis, Candida albicans, Escherichia coli, Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio vulnificus. All reagents and substrates in this experiment were prepared in binding buffer. The culture medium is LB liquid medium. Experimental steps:

Step 1: After resuscitating the microorganisms preserved in our laboratory, culture them overnight in 5ml LB liquid medium at 37°C (28°C for Candida albicans) at 150rpm.

Step 2: Centrifuge the bacterial liquid (5000×g*5min) to collect the bacterial cells, then use TBS buffer (24.8mM Tris, 50mM NaCl, 2.7mM KCl, pH 7.4) to wash the bacterial cells for 3 times, and add 1ml of recombinant protein (concentration 1mg/ml), incubated at room temperature for 1h.

Step 3: Centrifuge the bacterial solution (5000×g*5min) to collect the bacterial cells, then wash the bacterial cells with TBS buffer solution for 3 times, add 200 μl of TBS buffer solution containing 12% SDS, and shake gently for 15 minutes.

Step 4: Take 40 μl of the shaken sample and prepare for electrophoresis, which is the protein sample eluted by SDS. The remaining samples were washed 3 times with TBS buffer, and then dissolved with 100 μl of TBS buffer, which was used as the bacterial protein sample after TBS elution.

Step 5: Perform Western blot analysis. The collected samples are subjected to 10% SDS-PAGE electrophoresis and the subsequent membrane transfer operation is carried out to transfer the target protein to the PVDF membrane. The transformed PVDF membrane was incubated in TBST blocking solution containing 5% skim milk for 3 hours at room temperature, and washed three times with TBST. Dilute the mouse anti-GST antibody (primary antibody) with TBST at a volume dilution ratio of 1:2000, immerse the washed PVDF membrane, and incubate overnight at 4°C. After re-washing the PVDF membrane, it was incubated with horseradish peroxidase (HRP) goat anti-mouse IgG at a volume dilution ratio of 1:4000 for 3 hours at room temperature, and finally developed using the G:BOX system of British Syngene Company.

The experimental results are shown in Figure 6. It can be observed that the recombinant serine protease inhibitor has weak binding activity with Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Vibrio parahaemolyticus, and has strong binding activity with Candida albicans and Vibrio vulnificus , no binding activity was detected with Vibrio alginolyticus.

Example 7: Detection of the ability of recombinant jellyfish serine protease inhibitors to inhibit microbial growth

After the relatively single target protein was obtained by the affinity chromatography method in Example 4, the microorganisms used in the microorganism growth inhibition experiment were: Staphylococcus aureus, Bacillus subtilis, Candida albicans, Escherichia coli and Vibrio vulnificus. All reagents and substrates in this experiment were prepared in binding buffer. The culture medium is LB liquid medium. Experimental steps:

Step 1: After resuscitating the microorganisms preserved in our laboratory, culture them overnight in 5ml LB liquid medium at 37°C (28°C for Candida albicans) at 150rpm.

Step 2: Dilute the overnight cultured microorganisms 100 times with LB medium, then add serine protease inhibitor samples (concentrations are 0, 0.25, 0.5, 1 mg/ml respectively), and culture in a 96-well plate for 48 hours. Absorbance values were measured at 595 nm every 1 hour. Three replicate holes were made in parallel for each group.

The third step: data processing, according to the detection principle of the microplate reader, the higher the bacterial concentration, the higher the absorbance value. The concentration of the inhibitor was 0mg/ml as the negative control group, and the others were used as the treatment group to compare the trend of microbial growth.

The experimental results are shown in Figure 7, with the increase of the concentration of the recombinant serine protease inhibitor, its growth inhibition on Staphylococcus aureus, Bacillus subtilis, Candida albicans, Escherichia coli and Vibrio vulnificus increased to varying degrees.

The above results show that the recombinant jellyfish serine protease inhibitor of the present invention has significant serine protease inhibitory activity and antibacterial activity, and can be used for the development of antibacterial drugs, anti-inflammatory drugs, skin protective agents and the like.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments, and that described in the above-mentioned embodiments and the description only illustrates the principles of the present invention, and the present invention also has various aspects without departing from the spirit and scope of the present invention. Variations and improvements all fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (11)

1. A kind of 1. Cyanea capillata serpin, it is characterised in that described Cyanea capillata serine stretch protein Enzyme inhibitor is such as SEQ ID NO:The protein of amino acid sequence composition shown in 2.
2. A kind of 2. Cyanea capillata serpin according to claim 1, it is characterised in that：Described hair Shape rosy clouds jellyfish serpin contains signal peptide, is secretory protein, is positioned at extracellular, molecular weight 19.02kDa, Isoelectric point is 8.75.
3. A kind of 3. encoding gene of Cyanea capillata serpin as claimed in claim 1, it is characterised in that The nucleotide sequence of the encoding gene such as SEQ ID NO:Shown in 1.
4. 4. a kind of encoding gene of Cyanea capillata serpin according to claim 3, its feature exist In：The encoding gene of described Cyanea capillata serpin includes 531 bp ORFs.
5. A kind of 5. preparation method of Cyanea capillata serpin as claimed in claim 2, it is characterised in that This method comprises the following steps：

   (A) Cyanea capillata tentacle tissue cDNA library is built；

   (B) sequence of above-mentioned Cyanea capillata tentacle tissue cDNA library recombinant clone is measured and analyzed, obtain right It is required that the nucleotide sequence of the coding Cyanea capillata serpin described in 3；Using PCR method from hair shape Xia Shui The sequence is expanded in female tentacle tissue cDNA library；

   (C) expression plasmid of Cyanea capillata serpin, recombination engineering are built；

   (D) expression of Cyanea capillata serpin；

   (E) purifying of Cyanea capillata serpin is recombinated.
6. 6. the preparation method of Cyanea capillata serpin according to claim 5, it is characterised in that：Institute The Cyanea capillata tentacle tissue cDNA library stated in step (A) is built by the following method：

   A) Cyanea capillata tentacle total tissue RNA is extracted；

   B) mRNA is separated, synthesizes Cyanea capillata tentacle tissue cDNA；

   C) above-mentioned cDNA is inserted into pUC19 plasmid vectors, then converted into bacillus coli DH 5 alpha, be coated on LB culture medium flat plates and enter The blue hickie screening of row, that is, be built into Cyanea capillata tentacle tissue cDNA library.
7. 7. the preparation method of Cyanea capillata serpin according to claim 5, it is characterised in that：Institute State the expression plasmid of the structure Cyanea capillata serpin in step (C), recombination engineering, specific steps For：

   A) it is as follows to design and synthesize PCR primer：

   Sense primer such as SEQ ID NO:Shown in 3,

   Anti-sense primer such as SEQ ID NO:Shown in 4,

   PCR reaction conditions are：95 DEG C of insulation 30sec；95 DEG C insulation 30sec, 55.5 DEG C insulation 30sec, 68 DEG C insulation 1min, 38 Individual circulation；68 DEG C of insulation 5min；

   B) PCR primer and pGEX-6P-1 prokaryotic expression plasmids after digestion recovery；

   C) coded sequence is connected on pGEX-6P-1 carriers using two restriction enzyme sites, builds recombinant expression plasmid, Ran Houzhuan Change to Escherichia coli Rosetta (DE3) pLysS and obtain recombination engineering.
8. 8. the preparation method of Cyanea capillata serpin according to claim 5, it is characterised in that：Institute The expression condition for stating the Cyanea capillata serpin in step (D) is：12 DEG C, 1mM IPTG, 150rpm rings Induced 7 hours in border.
9. 9. the preparation method of Cyanea capillata serpin according to claim 5, it is characterised in that：Institute State the purifying in step (E) to produce to purify using the step of GSTrap HP 4B FF affinity columns one, elution requirement is to account for respectively The combination buffer of cumulative volume 90% and 10% elution buffer；Wherein, combination buffer is 1.8mM KH2PO4,140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, pH 7.3, elution buffer are 50mM Tris-HCl, 10mM reduced form gluathiones Peptide, pH 8.0.
10. 10. a kind of Cyanea capillata serpin as claimed in claim 1 or 2 is preparing antibacterials, disappeared Application in scorching medicine or shielding medicine for skin.
11. 11. a kind of encoding gene of Cyanea capillata serpin as claimed in claim 3 is preparing antibacterial Application in medicine, anti-inflammation drugs or shielding medicine for skin.