Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a collagenase mutant, a gene fragment, a recombinant plasmid, a recombinant expression system, a collagen peptide, a preparation method and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a collagenase mutant, the amino acid sequence of which has at least 99% sequence identity with the sequence shown as SEQ ID NO. 5.
Further, the amino acid sequence of the enzymatic collagenase mutant has at least 99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8% or at least 99.9% identity with the sequence as set forth in SEQ ID NO. 5.
Further, the enzyme collagenase mutant has an amino acid sequence shown in SEQ ID NO. 5.
The invention also provides a gene fragment for encoding the collagenase mutant, and the nucleotide sequence of the gene fragment is shown as SEQ ID NO. 7.
The invention also provides a recombinant plasmid, which comprises the gene fragment.
Further, the plasmid is a plasmid obtained by inserting the above gene fragment into pTR50 vector.
The invention also provides a recombinant expression system which contains host bacteria and the recombinant plasmid.
Further, the host bacterium of the recombinant expression system is Trichoderma reesei Trichoderma reeseiKH.
The invention also provides a method for preparing the collagenase mutant, which comprises the following steps:
(1) The sequence of expression cassette containing orotic acid phosphoribosyl transferase gene pyr2 as shown in SEQ ID NO. 1, the sequence segment containing promoter, signal peptide, polyclonal site and terminator as shown in SEQ ID NO. 2, and the sequence segment containing escherichia coli replicon and ampicillin resistance gene as shown in SEQ ID NO. 3 are subjected to seamless cloning to obtain vector pTR50;
(2) Cloning the DNA fragment shown in SEQ ID NO. 7 into a vector pTR50, and transforming escherichia coli DH5 alpha to obtain a plasmid pT513;
(3) Transferring plasmid pT513 into Trichoderma reesei Trichoderma reeseiKH, collecting bacterial colony spores, culturing, and collecting fermentation supernatant enzyme solution to obtain collagenase mutant.
The invention also provides application of the collagenase mutant, the gene fragment, the recombinant plasmid and the recombinant expression system in preparing collagen peptide.
The invention also provides a collagen peptide, which is a product obtained by taking collagen as a raw material and performing enzymolysis on the collagenase mutant.
Further, the collagen is fish scale collagen.
Further, the fish scale collagen is red snapper fish scale collagen.
The invention also provides a method for preparing the collagen peptide, which comprises the following steps of taking collagen as a raw material, and carrying out enzymolysis on the collagen peptide by using the collagenase mutant.
Further, the temperature of the enzymolysis is 40 ℃, the pH of the enzymolysis is 3.0, and the enzymolysis time is 4 h.
The invention also provides application of the collagen peptide in preparation of an antioxidant preparation.
The invention has the following beneficial effects:
According to the invention, a collagenase mutant KT513 with high specific activity is obtained through recombinant expression in a Trichoderma reesei host, and the enzyme activity of the collagenase mutant KT513 reaches 7423.18U/mL. The enzyme has an optimal pH of 3.0, can be used for enzymolysis of red snapper fish scale collagen within the pH range of 3.0-5.0, and has excellent enzymolysis activity. After enzymolysis of 4h at 40 ℃ by utilizing protease mutant KT513, the extraction rate of the fish scale collagen reaches 80.09%, and the prepared collagen peptide has good antioxidant activity. When the concentration of the collagen peptide is 10 mg/mL, the in-vitro scavenging activity of the prepared collagen peptide on hydroxyl radicals reaches 99.26%, which is equivalent to that of ascorbic acid, and the collagen peptide has wide application prospect in preparing antioxidant preparations.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
Example 1 Gene amplification and expression plasmid cloning
The cloning process of the basic plasmid pTR50 comprises the steps of chemically synthesizing a sequence containing a whey acid phosphoribosyl transferase gene pyr2 expression cassette shown as SEQ ID NO. 1, chemically synthesizing sequence fragments containing a promoter, a signal peptide, a polyclonal site and a terminator shown as SEQ ID NO. 2, chemically synthesizing sequence fragments containing an escherichia coli replicon and an ampicillin resistance gene shown as SEQ ID NO. 3, performing seamless cloning on the three DNA fragments by using an In-Fusion cloning kit of Takara company, and obtaining the vector pTR50 through sequence determination.
The construction process of the expression plasmid pT501 comprises cloning the DNA fragment shown In SEQ ID NO: 6 into a vector pTR50 linearized by restriction enzyme NheI using an In-Fusion cloning kit from Takara, transforming E.coli DH 5. Alpha. And sequencing to obtain the plasmid pT501.
The construction process of the expression plasmid pT513 comprises cloning the DNA fragment shown In SEQ ID NO: 7 into a vector pTR50 linearized by restriction enzyme NheI using an In-Fusion cloning kit from Takara, transforming E.coli DH 5. Alpha. And sequencing to obtain the plasmid pT513.
The DNA fragment shown in SEQ ID NO. 7 may be obtained by chemical synthesis and PCR amplification.
Example 2 heterologous expression of collagenases KT501 and KT513 and preparation of fermentation supernatant enzyme solution
The preparation method of collagenase KT501 and KT513 heterologous expression and fermentation supernatant enzyme liquid comprises the steps of transferring expression plasmids pT501 and pT513 provided in the previous example into protoplasts of uracil auxotropy host Trichoderma reesei Trichoderma reeseiKH respectively, screening positive colonies on a protoplast transformation regeneration medium (bottom layer medium: malt extract 30 g/L, sorbitol 1mol/L, agar powder 1.5%, upper layer medium: malt extract 30 g/L, sorbitol 1mol/L, agarose 0.7%) for 4-5 days, culturing at 30 ℃, picking possible positive colonies grown on a transformation plate to a MM basal medium solid plate without uridine addition, culturing at 30 ℃ for 2-3 days, screening a pyr2 gene expression cassette again to complement positive colonies integrated on a genome, inoculating colonies which normally grow on the multiplex screen plate to a PDA solid plate without uridine addition, culturing at 30 ℃ for 7-8 days to a green spore full plate.
Shake flask fermentation screening of recombinant strains spores of each colony were collected from PDA plates, diluted to 106 spores/mL, 500. Mu.L of spore suspension was transferred to 20 mL seed medium (glucose 2%, yeast powder 1%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.02%, pH 6.0), and cultured at 30℃and 220 r/min for 48 h. Transferring 2 mL seed solution into 50 mL fermentation medium (glucose 2%, tryptone 2%, dipotassium hydrogen phosphate 0.1%, potassium dihydrogen phosphate 0.1%, anhydrous calcium chloride 0.01%, magnesium sulfate 0.005%,1 mL microelements, microelements including manganese sulfate 0.16%, ferrous sulfate 0.2%, zinc sulfate 0.14%, and cobalt chloride 0.2%), culturing at 30deg.C and 220 r/min for 96 h. And collecting fermentation supernatant enzyme liquid, namely respectively obtaining fermentation supernatant enzyme liquid containing target protease KT501 and fermentation supernatant enzyme liquid containing target protease KT 513. The molecular weights of proteases KT501 and KT513 were analyzed by SDS-PAGE, and the results are shown in FIG. 1.
The amino acid sequence of the target protease KT513 is shown as SEQ ID NO. 5.
The following experiments prove the beneficial effects of the invention.
Experimental example 1 enzyme Activity measurement of mutant enzyme of the present invention under acidic conditions
The fermentation supernatant enzyme solutions containing the protease KT501 and the fermentation supernatant enzyme solution containing the protease KT513 obtained in example 2 were diluted to 10 to 15U/mL with the buffer solutions of the above pH of 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0, respectively, in the preparation of 0.1 mol/L Britton-Robinson buffer solution. The enzymatic activity of the proteases KT501, KT513 on the substrate casein was determined at 40℃and corresponding pH conditions. The highest enzyme activity is defined as 100%, the corresponding pH is the optimal action pH of the enzyme on casein, and the relative enzyme activities of other pH conditions are calculated.
Referring to GBT 23527-2009 protease preparation, spectrophotometry is used for measuring the enzyme activity of acid protease. Casein was used as a substrate, reacted in a 40 ℃ water bath for 10min, and quenched with trichloroacetic acid (TCA). After the precipitation was filtered, the reaction was developed with a furin reagent 20 min, and the absorbance was measured 680 to nm by a spectrophotometer.
The enzyme activity is defined as the amount of enzyme required to hydrolyze casein to produce 1 μg tyrosine per minute under specific conditions, i.e., 1 enzyme activity unit. Under the conditions of 40 ℃ and pH of 3.0, the highest expression enzyme activity of the enzyme KT501 is 4231.63U/mL, and the highest expression enzyme activity of the mutant enzyme KT513 is 7423.18U/mL, which is improved by 1.75 times.
As a result, as shown in FIG. 2, enzymes KT501 and KT513 each had casein degrading activity in the pH range of 2.0 to 7.0, and the optimum pH was 3.0, indicating that both enzymes were acid proteases. Wherein, the pH value is 3.0-5.0, KT501 keeps the relative enzyme activity of more than 65.77%, the mutant enzyme KT513 can keep the relative enzyme activity of more than 70.21%, and the relative enzyme activity is obviously improved compared with KT 501. The pH curve measurement result shows that the mutant enzyme KT513 has better applicability under the acidic condition.
The results show that compared with the enzyme KT501, the mutant enzyme KT513 has obviously improved enzyme activity and better applicability under acidic conditions.
Experimental example 2 pH stability of mutant enzyme of the present invention
The fermentation supernatant enzyme solution containing protease KT501 and the fermentation supernatant enzyme solution containing protease KT513 obtained in example 2 were diluted to 10 to 15U/mL with 0.1 mol/L Britton-Robinson buffer having pH of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and left to stand for 2h, and the residual enzyme activity at each pH condition was measured at 40℃and the enzyme activity of the raw fermentation supernatant enzyme solution without acid treatment was defined as 100%.
As a result, as shown in FIG. 3, the mutant enzyme KT513 exhibited higher stability than the original enzyme KT501 after 2 h treatment with a buffer solution having a pH of 3.0-6.0. The specific data are that after incubation of 2 h under the condition of pH 3.0, KT501 can keep 60.46% of residual enzyme activity, the relative enzyme activity of mutant enzyme KT513 reaches 70.19%, the stability is improved by 16.09%, after treatment of 2 h under the condition of pH 4.0-6.0, the activity of original enzyme KT501 remains 70.13% -81.50%, the relative enzyme activity of mutant enzyme KT513 is 76.17% -88.06%, which further proves that the stability of the mutant enzyme is obviously improved in the pH range of 3.0-6.0.
Experimental example 3 collagen enzymolysis Property of mutant enzyme of the invention
And (3) flushing the collected red snapper scales with flowing clear water to remove impurities such as sediment, mucus, blood and the like attached to the surfaces. Then, the fish scales are soaked in 5% (w/v) citric acid solution with the volume of 12 times, and decalcified by stirring at the temperature of 4 ℃, so that the fish scales are gradually transparent and soft. When the decalcification solution (i.e. the solution of fish scales reacted with 5% (w/v) citric acid solution) was mixed with the ammonium oxalate solution, there was no white precipitate anymore, indicating that decalcification was essentially complete. Taking the fish scales out of the citric acid solution, repeatedly flushing with flowing clean water to remove residual citric acid solution until the flushed water is neutral, and airing the fish scales.
Adding water with volume 4 times that of fish scales, stirring thoroughly, and adjusting pH of the mixed solution to 7.0-8.0. Adding lipase according to the weight ratio of 0.2 per mill of the fish skin, and continuously stirring and reacting at 30 ℃ for 2 h to finish degreasing the fish scales. After the degreasing reaction was completed, the temperature of the reaction solution was raised to 100℃and boiled to 20min to inactivate the lipase. The reaction solution was cooled to 45-50 ℃, the initial pH of the reaction solution was adjusted to 5.0 with acetic acid solution, and 1%o (w/w) of the fermentation supernatant enzyme solution containing protease KT501 and the fermentation supernatant enzyme solution containing protease KT513 obtained in example 2 were added respectively, and stirred and reacted at 40 ℃ to obtain an aqueous solution of collagen peptide of red snapper fish scales digested with acid proteases KT501 and KT 513. Through detection, the pH of the reaction solution is kept in the range of 3.0-5.0 in the enzymolysis process. Wherein, when reactions 1 h, 2 h, 3 h, 4h and 5 h are respectively carried out, the extraction rate of collagen in the enzymolysis liquid (also called reaction liquid) is measured, and the method for calculating the extraction rate of collagen is as follows:
Referring to GB 5009.5-2016 (determination of protein in national food safety Standard), the total nitrogen content in red sea bream fish scale collagen is determined by Kjeldahl nitrogen determination method. The mass concentration of the L-hydroxyproline standard substance is on the abscissa, the light absorption value at 560 nm is on the ordinate, a standard curve is drawn, the regression equation is y=0.4332x+0.0003 (R2 =0.99995), and the hydroxyproline content in fish scale collagen enzymolysis is measured and calculated. Collagen extraction was expressed in hydroxyproline concentration by the following formula.
Extraction yield (%) =m1 ×dilution x 11.1/m0 ×100
Wherein m1 is the hydroxyproline content, g, 11.1 is the coefficient of conversion of hydroxyproline into collagen, and m0 is the total amount of red snapper fish scale protein.
As can be seen from fig. 4, the collagen extraction rate increases with the increase of the enzymolysis time. After enzymolysis by the enzyme KT501 for 2h, the extraction rate of the collagen is 60.46%, the extraction rate is slowly increased after 3 h, and the extraction rate reaches 70.56% after 4 h. After 2h of enzyme KT513 is hydrolyzed, the extraction rate of the collagen is 67.21%, which is 111.16% of the control group enzyme KT501, and after 4h of enzyme KT513 is hydrolyzed, the extraction rate of the collagen reaches 80.90%, which is 114.65% of the control group enzyme KT 501.
The results show that compared with the enzyme KT501, the enzymatic hydrolysis capability of the mutant enzyme KT513 to collagen is obviously improved, and the collagen extraction rate is obviously improved.
Experimental example 4 determination of molecular weight of peptide liquid obtained after enzymatic hydrolysis of mutant enzyme of the present invention
The molecular weight distribution of peptide fragments in the collagen peptide aqueous solution of red snapper fish scales prepared by the enzymolysis of 4h by proteases KT501 and KT513 in experimental example 3 is detected by an HPLC method. 5 standards were selected, including cytochrome C (mr=12500 Da), aprotinin (mr=6500 Da), bacitracin (mr=1450 Da), tetrapeptide GGYR (mr=451 Da) and tripeptide GGG (mr=189 Da). The measurement was carried out using a TSK gel G2000 SWXL column (specification: 7.8X100 mm) produced by Tosoh corporation, japan, with a 45% acetonitrile solution containing 0.1% (v/v) trifluoroacetic acid as a mobile phase at a flow rate of 0.5 mL/min at a wavelength of 220 nm. And drawing a standard curve according to the peak time and the molecular weight of each chromatographic peak of the standard substance. The two peptide solutions prepared in the above examples were diluted to 10 mg/mL, subjected to 0.22 μm membrane filtration, 20. Mu.L was measured, and the molecular weight of the peptide fragment and the ratio of each component in the peptide solution prepared by enzymatic hydrolysis of the proteases KT501 and KT513 were calculated.
TABLE 1 molecular weight of peptide fragments in collagen peptide aqueous solution of fish scales of red porgy
The results are shown in Table 1, the aqueous solution of red snapper fish scale collagen peptide prepared by enzymolysis of original enzyme KT501 after enzymolysis of 4h takes peptide segment with molecular weight lower than 3000Da as main component, the ratio is 90.11%, wherein the content of peptide segment with molecular weight lower than 1000 Da is 68.88%. In the red snapper fish scale collagen peptide aqueous solution prepared by enzymolysis of mutant enzyme KT513, the ratio of peptide segments with the molecular weight lower than 3000Da reaches 92.03%, wherein the content of peptide segments with the molecular weight lower than 1000 Da is improved to 77.42%, and the ratio of peptide segments with the molecular weight lower than 500Da reaches 33.85%, which is 1.8 times that of the original enzyme KT 501. The experimental result shows that the mutant enzyme KT513 can prepare a high-content small molecular weight peptide fragment, which is more beneficial to keeping the better physiological activity of the collagen peptide.
Experimental example 5 in vitro hydroxyl radical scavenging Activity of peptide solution obtained after enzymatic hydrolysis of mutant enzyme of the invention
Hydroxyl radical (.OH) can trigger a series of diseases related to oxidative stress, and researches show that collagen peptide has antioxidant activity, especially has the function of scavenging the hydroxyl radical. The removal effect of collagen peptide in the collagen peptide aqueous solution of red snapper fish scales prepared by enzymolysis 4h of protease KT501 and KT513 in test example 3 on hydroxyl free radicals is detected by Fenton reaction.
The fish scale collagen peptide aqueous solution (also called enzymolysis peptide solution) of red snapper is prepared with concentration of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 mg/mL. 1mL of enzymolysis peptide liquid, 1mL of 9 mmol/L salicylic acid-ethanol solution, 1mL of 9 mmol/L ferrous sulfate solution and 1mL of 8.8 mmol/L H2O2 solution are sequentially added into a test tube to start the reaction. Incubation was performed in a 37 ℃ thermostat water bath at 30min and absorbance of the mixture was measured at 510 and nm. Under the same conditions, the same volume of distilled water is used for replacing 8.8 mmol/L H2O2 solution as a background group, the same volume of distilled water is used for replacing the sample as a blank control group, and the same volume of ascorbic acid is used for replacing the sample as a positive control group.
Radical scavenging rate was calculated using the following formula:
Clearance (%) = [1- (a1-A2) / A0 ] ×100)
Wherein a1 is the absorbance of the sample, a2 is the absorbance of the background group, and a0 is the absorbance of the blank group.
As can be seen from FIG. 5, the scavenging ability of the enzymatic hydrolysis peptide solution with concentration of 1-10 mg/mL to hydroxyl radical (. OH) increases with the increase of the solution concentration. The peptide liquid prepared by the enzymolysis of the original enzyme KT501 has the highest 86.31 percent of scavenging activity when the adding amount is 10 mg/mL. The peptide liquid prepared after enzymolysis of mutant enzyme KT513 has the scavenging activity of 99.26% when the addition amount is 10 mg/mL, which is equivalent to the scavenging ability of ascorbic acid. The experimental results show that compared with the peptide liquid prepared by the enzymolysis of the original enzyme KT501, the peptide liquid prepared by the enzymolysis of the mutant enzyme KT513 has obviously improved capability of scavenging the hydroxyl free radicals.
In conclusion, the collagenase mutant KT513 with high specific activity is obtained through recombinant expression in the Trichoderma reesei host, and the enzyme activity of the collagenase mutant KT513 reaches 7423.18U/mL. The enzyme has an optimal pH of 3.0, can be used for enzymolysis of red snapper fish scale collagen within the pH range of 3.0-5.0, and has excellent enzymolysis activity. After enzymolysis of 4h at 40 ℃ by utilizing protease mutant KT513, the extraction rate of the fish scale collagen reaches 80.09%, and the prepared collagen peptide has good antioxidant activity. When the concentration of the collagen peptide is 10 mg/mL, the in-vitro scavenging activity of the prepared collagen peptide on hydroxyl radicals reaches 99.26%, which is equivalent to that of ascorbic acid, and the collagen peptide has wide application prospect in preparing antioxidant preparations.
And (3) a sequence table:
SEQ ID NO: 1
aggtgggcgagatttatattgacggacgcaacatcaaagcgttggacaagaagtcgtatcgcagtcatttggcgctggtcagtcaggaaccatcgctgttccatggcactatccgggagaacattctcctgggttgtacggataaggaacatgtgtcggaggatatggtggtcagagcgtgcagagatgcgaatatttatgatttcatcatgtcgttgccgtgagtcctccctgtccctttcctctttgtggtatatatgcggttactgatagagaaacagacaaggctttgacaccctcgttggtaacaagggtggcatgttgtcaggtggacagaaacagcgtatcgcgattgcgcgtgcgttgattcggaacccgcgcattttgttgttggacgaggcgacttccgcgctggattctgagtcggagaaggtggtgcaggctgcgctggacgcggctgccaaggggaggaccactattgcggtggcgcatcggctaagtacgattcaacgggcggatatgatctatttcttagagcagggagaggtgattgagtgtgggacacattaggagctgttgaggaggaggggacggtattatgagatggtgaatttgcagactttgaggtgatgataccattgacttggtgggtggttcatgggttatgtgaaggcgttagtggtaatgtatattaatggtgagatgggctttgattgggtttaattggaatctgtatattttcagatggagtcaacttttgaatggcaatatatcctcggcgataccgtcggagataagataagaataatcgcacactattcccaaagcatactggtacatactgcattcggctagtgcggggtgcttacctcatccacccgaatgagcccaacttttttgtctcaatcaataattgcatccaaattcccccgcaacttccccctccaaccccgtgtctataccactccctccacacccacacaatcacaatgagcgacatggagaagccctggaaggagggcgaggaggcccgagccgtcctccagggccacgctcgcgcccaggagcctcaggccgtcgacaagggccccgtggccggcgacgagcgcatggccgtcaccgtcgtcctccgccgccagcgcgccgacgctctcgccgctcacgtcgagcgccaggccgccattgctccccacgcgcgcgagcacctcaagcgcgaggccttcgccgcctcgcacggcgccagcctcgacgacttcgccgagctgcgccgcttcgctgacgcccacggcctcgctctcgaccgcgccaacgtcgccgctggcaccgccgtcctcagcggccctgtggacgccatcaaccgcgccttcggcgtcgagctgcgacacttcgatcaccccgacggcagctaccgctcctacctcggcgaggtcacggtccccgcctcgatcgcccctatgatcgaggccgtcctcggcctcgacactcgccccgtcgctcgcccgcacttccgcatgcagcgccgagccgagggcggcttcgaggcccgctcgcaggccgctgctcccaccgcctacacgcccctcgacgtcgcccaggcctatcagttccccgagggcctcgacggccagggccagtgcattgccatcatcgagcttggcggcggctacgacgaggcctctctggcccagtacttcgccagcctgggcgtccccgctcctcaggtcgtcagcgtcagcgtggacggcgcctccaaccagcctacgggcgacccttcgggccctgatggcgaggtcgagctggatatcgaggtcgctggcgccctcgctcctggcgccaagatcgccgtctacttcgcccctaacaccgacgccggctttctcgacgccattaccaccgccattcacgaccccacgctcaagcccagcgtcgtcagcatctcctggggcggccccgaggacagctggcccagcgccgctattgccgctatgaaccgagccttcctcgatgccgccgctctgggcgtcaccgtgctcgccgctgccggcgactccggcagcaccgacggcgagcaggatggcctctaccacgtcgacttccccgctgctagcccctacgtcctcgcctgcggcggcacccgcctggtggctagcggcggcaggattgcccaggagactgtctggaacgacggccccgatggcggcgctaccggcggcggcgtcagccgcatcttccctctgcctgcctggcaggagcacgctaacgtccctccgagcgctaaccctggcgcctcctccggccgaggcgtccctgacctggccggcaacgctgaccccgccaccggctacgaggtcgtcattgacggcgaggccaccgtcatcggcggcacgagcgccgtcgcgcccctcttcgctgccctcgtcgcccgcatcaaccagaagctcggcaaggccgtcggctatctcaaccccactctctaccagctgcctgccgacgtctttcacgacatcaccgagggcaacaacgacattgccaaccgcgctcagatctatcaggctggccctggctgggacccctgcaccggcctcggcagccctatcggcgtccgcctcctgcaggccctcctgcctagcgcctcgcagccccagccttcggtttcattgaccgattgtttgggtgggtgtgagaggttaggttaggttgtgggcgtaggaatgaaaagctgtatacataggggcctgaagaggtgcgtagagacggtcgtgagatgttttatgtcaaaatcttgaacaaatgacaccttaaaaaagaccccttggtttcagctgaattagcccggaaagatgctcggcacgccatgagtctagcccactcagtgggcacccgtttcccacatttgaagtggccgacgcttat
SEQ ID NO: 2
tctagagttgtgaagtcggtaatcccgctgtatagtaatacgagtcgcatctaaatactccgaagctgctgcgaacccggagaatcgagatgtgctggaaagcttctagcgagcggctaaattagcatgaaaggctatgagaaattctggagacggcttgttgaatcatggcgttccattcttcgacaagcaaagcgttccgtcgcagtagcaggcactcattcccgaaaaaactcggagattcctaagtagcgatggaaccggaataatataataggcaatacattgagttgcctcgacggttgcaatgcaggggtactgagcttggacataactgttccgtaccccacctcttctcaacctttggcgtttccctgattcagcgtacccgtacaagtcgtaatcactattaacccagactgaccggacgtgttttgcccttcatttggagaaataatgtcattgcgatgtgtaatttgcctgcttgaccgactggggctgttcgaagcccgaatgtaggattgttatccgaactctgctcgtagaggcatgttgtgaatctgtgtcgggcaggacacgcctcgaaggttcacggcaagggaaaccaccgatagcagtgtctagtagcaacctgtaaagccgcaatgcagcatcactggaaaatacaaaccaatggctaaaagtacataagttaatgcctaaagaagtcatataccagcggctaataattgtacaatcaagtggctaaacgtaccgtaatttgccaacggcttgtggggttgcagaagcaacggcaaagccccacttccccacgtttgtttcttcactcagtccaatctcagctggtgatcccccaattgggtcgcttgtttgttccggtgaagtgaaagaagacagaggtaagaatgtctgactcggagcgttttgcatacaaccaagggcagtgatggaagacagtgaaatgttgacattcaaggagtatttagccagggatgcttgagtgtatcgtgtaaggaggtttgtctgccgatacgacgaatactgtatagtcacttctgatgaagtggtccatattgaaatgtaagtcggcactgaacaggcaaaagattgagttgaaactgcctaagatctcgggccctcgggccttcggcctttgggtgtacatgtttgtgctccgggcaaatgcaaagtgtggtaggatcgaacacactgctgcctttaccaagcagctgagggtatgtgataggcaaatgttcaggggccactgcatggtttcgaatagaaagagaagcttagccaagaacaatagccgataaagatagcctcattaaacggaatgagctagtaggcaaagtcagcgaatgtgtatatataaaggttcgaggtccgtgcctccctcatgctctccccatctactcatcaactcagatcctccaggagacttgtacaccatcttttgaggcacagaaacccaatagtcaaccgcggactgcgcaccatgtatcggaagttggccgtcatctcggccttcttggccacagctcgtgctagctccgtggcgaaagcctgacgcaccggtagattcttggtgagcccgtatcatgacggcggcgggagctacatggccccgggtgatttattttttttgtatctacttctgacccttttcaaatatacggtcaactcatctttcactggagatgcggcctgcttggtattgcgatgttgtcagcttggcaaattgtggctttcgaaaacacaaaacgattccttagtagccatgcattttaagataacggaatagaagaaagaggaaattaaaaaaaaaaaaaaaacaaacatcccgttcataacccgtagaatcgccgctcttcgtgtatcccagtaccactcgag
SEQ ID NO: 3
ggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcccttt
SEQ ID NO: 4
msdmekpwkegeearavlqgharaqepqavdkgpvagdermavtvvlrrqradalaahverqaaiapharehlkreafaashgaslddfaelrrfadahglaldranvaagtavlsgpvdainrafgvelrhfdhpdgsyrsylgevtvpasiapmieavlgldtrpvarphfrmqrraeggfearsqaaaptaytpldvaqayqfpegldgqgqciaiielgggydeaslaqyfaslgvpapqvvsvsvdgasnqptgdpsgpdgeveldievagalapgakiavyfapntdagfldaittaihdptlkpsvvsiswggpedswpsaaiaamnrafldaaalgvtvlaaagdsgstdgeqdglyhvdfpaaspyvlacggtrlvasggriaqetvwndgpdggatgggvsrifplpawqehanvppsanpgassgrgvpdlagnadpatgyevvidgeatviggtsavaplfaalvarinqklgkavgylnptlyqlpadvfhditegnndianraqiyqagpgwdpctglgspigvrllqallpsasqpqp
SEQ ID NO: 5
msdmekpwkegeearavlqgharaqepqavdkgpvagdermavtvvlrrqradalaahverqaaiapharehlkreafaashgaslddfaelrrfadahglaldranvaagtavlsgpvdainrafgvelrhfdhpdgsyrsylgevtvpasiapmieavlgldtrpvarphfrmqrraeggfearsqaaaptaytpldvaqayqfpegldgqgqciaiielgggydeaslaqyfaslgvpapqvvsvsvdgasnqptgdpegpdghvtldievagalapgakiavyfapdttagfldaittaihdptlkpsvvsiswggpedswpsaaiaamnrafldaaalgvtvlaaagnqgstsgeqdglyhvdfpaaspyvlacggtrlvasggriaqetvwndgpdggatgggvsrifplpawqehanvppsanpgassgrgvpdlagnadpatgyevvidgeatviggtsavaplfaalvarinqklgkavgylnptlyqlpadvfhditegnndianraqiyqagpgwdpctglgspigvrllqallpsasqpqp
SEQ ID NO: 6
atgagcgacatggagaagccctggaaggagggcgaggaggcccgagccgtcctccagggccacgctcgcgcccaggagcctcaggccgtcgacaagggccccgtggccggcgacgagcgcatggccgtcaccgtcgtcctccgccgccagcgcgccgacgctctcgccgctcacgtcgagcgccaggccgccattgctccccacgcgcgcgagcacctcaagcgcgaggccttcgccgcctcgcacggcgccagcctcgacgacttcgccgagctgcgccgcttcgctgacgcccacggcctcgctctcgaccgcgccaacgtcgccgctggcaccgccgtcctcagcggccctgtggacgccatcaaccgcgccttcggcgtcgagctgcgacacttcgatcaccccgacggcagctaccgctcctacctcggcgaggtcacggtccccgcctcgatcgcccctatgatcgaggccgtcctcggcctcgacactcgccccgtcgctcgcccgcacttccgcatgcagcgccgagccgagggcggcttcgaggcccgctcgcaggccgctgctcccaccgcctacacgcccctcgacgtcgcccaggcctatcagttccccgagggcctcgacggccagggccagtgcattgccatcatcgagcttggcggcggctacgacgaggcctctctggcccagtacttcgccagcctgggcgtccccgctcctcaggtcgtcagcgtcagcgtggacggcgcctccaaccagcctacgggcgacccttcgggccctgatggcgaggtcgagctggatatcgaggtcgctggcgccctcgctcctggcgccaagatcgccgtctacttcgcccctaacaccgacgccggctttctcgacgccattaccaccgccattcacgaccccacgctcaagcccagcgtcgtcagcatctcctggggcggccccgaggacagctggcccagcgccgctattgccgctatgaaccgagccttcctcgatgccgccgctctgggcgtcaccgtgctcgccgctgccggcgactccggcagcaccgacggcgagcaggatggcctctaccacgtcgacttccccgctgctagcccctacgtcctcgcctgcggcggcacccgcctggtggctagcggcggcaggattgcccaggagactgtctggaacgacggccccgatggcggcgctaccggcggcggcgtcagccgcatcttccctctgcctgcctggcaggagcacgctaacgtccctccgagcgctaaccctggcgcctcctccggccgaggcgtccctgacctggccggcaacgctgaccccgccaccggctacgaggtcgtcattgacggcgaggccaccgtcatcggcggcacgagcgccgtcgcgcccctcttcgctgccctcgtcgcccgcatcaaccagaagctcggcaaggccgtcggctatctcaaccccactctctaccagctgcctgccgacgtctttcacgacatcaccgagggcaacaacgacattgccaaccgcgctcagatctatcaggctggccctggctgggacccctgcaccggcctcggcagccctatcggcgtccgcctcctgcaggccctcctgcctagcgcctcgcagccccagcct
SEQ ID NO: 7
atgagcgacatggagaagccctggaaggagggcgaggaggcccgagccgtcctccagggccacgctcgcgcccaggagcctcaggccgtcgacaagggccccgtggccggcgacgagcgcatggccgtcaccgtcgtcctccgccgccagcgcgccgacgctctcgccgctcacgtcgagcgccaggccgccattgctccccacgcgcgcgagcacctcaagcgcgaggccttcgccgcctcgcacggcgccagcctcgacgacttcgccgagctgcgccgcttcgctgacgcccacggcctcgctctcgaccgcgccaacgtcgccgctggcaccgccgtcctcagcggccctgtggacgccatcaaccgcgccttcggcgtcgagctgcgacacttcgatcaccccgacggcagctaccgctcctacctcggcgaggtcacggtccccgcctcgatcgcccctatgatcgaggccgtcctcggcctcgacactcgccccgtcgctcgcccgcacttccgcatgcagcgccgagccgagggcggcttcgaggcccgctcgcaggccgctgctcccaccgcctacacgcccctcgacgtcgcccaggcctatcagttccccgagggcctcgacggccagggccagtgcattgccatcatcgagcttggcggcggctacgacgaggcctctctggcccagtacttcgccagcctgggcgtccccgctcctcaggtcgtcagcgtcagcgtggacggcgcctccaaccagcctacgggcgaccctgagggccctgatggccacgtcaccctcgacatcgaggtcgctggcgccctcgctcctggcgccaagatcgccgtctacttcgcccctgatacgaccgccggcttcctcgacgccattaccaccgccattcacgaccccacgctcaagcccagcgtcgtcagcatctcctggggcggccccgaggacagctggcccagcgccgctattgccgctatgaaccgagccttcctcgatgccgccgctctgggcgtcaccgtgctcgccgctgccggcaaccagggcagcacctccggcgagcaggatggcctctaccacgtcgacttccccgctgctagcccctacgtcctcgcctgcggcggcacccgcctggtggctagcggcggcaggattgcccaggagactgtctggaacgacggccccgatggcggcgctaccggcggcggcgtcagccgcatcttccctctgcctgcctggcaggagcacgctaacgtccctccgagcgctaaccctggcgcctcctccggccgaggcgtccctgacctggccggcaacgctgaccccgccaccggctacgaggtcgtcatcgacggcgaggccaccgtcatcggcggcacgagcgccgtcgcgcccctcttcgctgccctcgtcgcccgcatcaaccagaagctcggcaaggccgtcggctatctcaaccccactctctaccagctgcctgccgacgtctttcacgacatcaccgagggcaacaacgacattgccaaccgcgctcagatctatcaggctggccctggctgggacccctgcaccggcctcggcagccctatcggcgtccgcctcctgcaggccctcctgcctagcgcctcgcagccccagcct .