Recombinant chondrosulphatase AC and preparation method and application thereof
1. A recombinant chondroitinase AC is characterized in that the nucleotide sequence of the recombinant chondroitinase AC is shown in SEQ ID NO. 3, the full length of the gene is 2097 bp, 701 amino acids are coded, and the molecular weight is 77 kDa.
2. The recombinant chondrosulphatase AC according to claim 1, wherein the amino acid sequence is as shown in SEQ ID NO 4.
3. A preparation method of recombinant chondroitinase AC is characterized by comprising the following steps:
(1) taking a chondroitinase AC genome of Pedobacter sp. ok626 as a template, adopting enzyme cutting sites with Bam HI and Xho I and 5' -end protection bases as primers, and carrying out PCR amplification coding according to a conventional method to obtain a target gene segment;
(2) connecting the target gene fragment with an expression vector pET28a to construct a pET-28a-ChSaSeAC recombinant expression vector of the chondroitinase sulfate AC gene;
(3) transforming the pET-28a-ChSaSeAC recombinant expression vector constructed in the step (2) into escherichia coli host bacteria to construct chondrosulphatase AC gene escherichia coli engineering bacteria;
(4) induced expression of recombinant chondroitinase in chondroitinase AC gene escherichia coli engineering bacteria;
(5) and (5) separating and purifying the expression product obtained in the step (4) to obtain the recombinant chondroitinase AC protein.
4. The method for constructing a recombinant Human Immunodeficiency Virus (HIV) vaccine according to claim 3, wherein the sequence of the upstream primer of the primer in the step (1) is as shown in SEQ ID NO:1 is shown in the specification; the sequence of the downstream primer is shown as SEQ ID NO:2, respectively.
5. The method of claim 3, wherein the reaction parameters of the PCR amplification in step (1) are: initial denaturation at 95 deg.C for 4 min, denaturation at 95 deg.C for 30 s, annealing at 60 deg.C for 55 s, extension at 72 deg.C for 1 min, circulation for 20 times, and extension at 72 deg.C for 10 min.
6. The method for constructing a recombinant plasmid of claim 3, wherein the target gene fragment and the expression vector pET28a in step (2) are subjected to double digestion treatment by using BamHI and Xho I, respectively.
7. The method of claim 3, wherein the ligation in step (2) is carried out at 25 ℃ for 12 hours; the connecting system is as follows: 2. mu.L of vector, 6. mu.L of target gene, 1. mu.L of 10 × Ligation buffer, and 1. mu.L of T4DNA ligase.
8. The method of claim 3, wherein the inducing expression in step (4) is: inoculating the chondroitinase AC gene engineering bacterium to 5 mL LB liquid culture medium containing 50 ug/mL kanamycin sulfate, and performing shaking culture at 200 rpm to OD600After reaching 0.7, IPTG was added to a final concentration of 0.1mM and shaken at 25 ℃ for 15 h.
9. The method according to claim 3, wherein the molecular weight of the recombinant chondroitinase ABC-I protein of step (5) is 77 kDa.
10. Use of the recombinant chondroitinase AC as claimed in claim 1 for the preparation of low molecular weight chondroitin sulfate.
Background art:
chondroitin sulfate (chondroitin sulfate) is a linear, sulfated, acidic polysaccharide composed of repeating disaccharide units formed by connecting uronic acid (L-iduronic acid or D-glucuronic acid) and galactosamine through 1 → 3 glycosidic linkages, and has an average molecular weight of about 25 kDa. Chondroitin sulfate mainly comprises three types of chondroitin sulfate A, chondroitin sulfate C and dermatan sulfate. It is widely found in a variety of animal tissues, and is particularly abundant in cartilage and connective tissue. Chondroitin sulfate has important effects on reducing blood fat, resisting atherosclerosis, protecting eye cornea and the like, and is widely used as an additive in high-grade health care products, foods, beverages, tonics and high-grade cosmetics. The application of the traditional Chinese medicine is limited due to the defects of large molecular mass fluctuation, low bioavailability, poor oral effect, unstable curative effect and the like. Many researches show that the low molecular weight chondroitin sulfate (the average molecular weight is 2-10 kDa) prepared by a chemical method or an enzymatic method can effectively overcome the defects.
Chondrosulphatase (ChSase) is a kind of lyase capable of degrading glycosaminoglycans such as chondroitin sulphate, chondroitin and hyaluronic acid into unsaturated disaccharides (Delta Di and oligosaccharides), which cleaves galactosamine beta (1 → 4) uronic acid glycosidic bond by beta-elimination mechanism and forms double bonds at 4, 5-positions of uronic acid, and is mainly classified into ChSae ABC, ChSase AC, ChSase B and ChSase C according to the difference of acting substrates. With the increasing international attention on ChSase AC, a preparation process method capable of obtaining a large amount of cheap ChSase AC is urgently needed. At present, the ChSase AC is obtained mainly through chondroitin sulfate flavobacteriumFlavobacterium heparinumObtained by fermentation and purification, but the chondroitin sulfate flavobacterium grows slowly, the ChSase AC expression level is low, the separation and purification are complex, and the specific activity is only 18.7U mg-1Therefore, the production cost of ChSase AC is directly increased, and the lower production capacity and the overhigh price are important factors for restricting the functional research and the application development of the ChSase AC.
Disclosure of Invention
In view of the above, the present invention provides a recombinant chondroitinase AC, and a preparation method and applications thereof. The recombinant chondroitin sulfate enzyme AC obtained by the molecular cloning method has higher enzyme activity and stability, simple preparation method and low cost, and can be used for industrially producing low-molecular-weight chondroitin sulfate.
The scheme of the invention is realized by the following technical means:
the invention provides a recombinant chondroitin sulfate enzyme AC, the nucleotide sequence of which is shown as SEQ ID NO. 3, the full length of the gene is 2097 bp, 701 amino acids are coded, and the molecular weight is 77 kDa.
In another aspect, the present invention also provides an amino acid sequence encoding the above chondroitinase AC, wherein the amino acid sequence is shown in SEQ ID NO. 4.
The invention also provides a preparation method of the recombinant chondroitinase AC, which comprises the following steps:
(1) taking a chondroitinase AC genome of Pedobacter sp. ok626 as a template, adopting enzyme cutting sites with Bam HI and Xho I and 5' -end protection bases as primers, and carrying out PCR amplification coding according to a conventional method to obtain a target gene segment;
(2) connecting the target gene fragment with an expression vector pET28a to construct a pET-28a-ChSaSeAC recombinant expression vector of the chondroitinase sulfate AC gene;
(3) transforming the pET-28a-ChSaSeAC recombinant expression vector constructed in the step (2) into escherichia coli host bacteria to construct chondrosulphatase AC gene escherichia coli engineering bacteria;
(4) induced expression of recombinant chondroitinase in chondroitinase AC gene escherichia coli engineering bacteria;
(5) and (5) separating and purifying the expression product obtained in the step (4) to obtain the recombinant chondroitinase AC protein.
Further, the sequence of the upstream primer of the primer in the step (1) is shown as SEQ ID NO:1 is shown in the specification; the sequence of the downstream primer is shown as SEQ ID NO:2, respectively.
The reaction parameters of the PCR amplification in the step (1) are as follows: initial denaturation at 95 deg.C for 4 min, denaturation at 95 deg.C for 30 s, annealing at 60 deg.C for 55 s, extension at 72 deg.C for 1 min, circulation for 20 times, and extension at 72 deg.C for 10 min.
And (3) carrying out double enzyme digestion treatment on the target gene fragment and the expression vector pET28a in the step (2) by using Bam HI and Xho I respectively.
The connection condition in the step (2) is 25 ℃ and 12 hours; the connecting system is as follows: 2. mu.L of vector, 6. mu.L of target gene, 1. mu.L of 10 × Ligation buffer, and 1. mu.L of T4DNA ligase.
The induced expression in the step (4) is as follows: inoculating the chondroitinase AC gene engineering bacterium to 5 mL LB liquid culture medium containing 50 ug/mL kanamycin sulfate, and performing shaking culture at 200 rpm to OD600After reaching 0.7, IPTG was added to a final concentration of 0.1mM and shaken at 25 ℃ for 15 h.
The molecular weight of the recombinant chondroitin sulfate enzyme AC in the step (5) is 77 kDa.
The invention also provides application of the recombinant chondroitin sulfate enzyme AC in preparation of low-molecular-weight chondroitin sulfate.
Compared with the prior art, the invention has the beneficial effects that:
the invention uses molecular cloning technology to design primers by extracting chondroitinase AC gene, and the primers are connected with an expression vector after PCR amplification to construct a recombinant expression vector; and transforming the recombinant expression vector to escherichia coli to construct a recombinant expression transformant, so as to realize the soluble expression of the recombinant chondroitinase ABC-I protein. The construction process is simple to operate and low in cost. The constructed recombinant chondroitinase ABC-I protein has high expression level and high enzyme activity, and the activity of crude enzyme can reach 5082U/L. The enzyme activity is 325.2.6U mg-protein,K mthe value was 0.5mg mL-1, V maxIs 509.43U mg-1. And the stability is good, the half-life period at 37 ℃ is 11 h, and the half-life period at 4 ℃ is 28 days. Can be used for preparing low molecular weight chondroitin sulfate, degrading the chondroitin sulfate in the heparin raw material and achieving the purpose of removing the chondroitin sulfate in the heparinoid raw material. Has important scientific significance for improving the technical level of biological expression of ChSase AC in China and expanding the application field of ChSase AC.
Drawings
FIG. 1 is a diagram of a double-restriction agarose electrophoresis analysis of a recombinant plasmid;
FIG. 2 is an electrophoretic analysis chart of recombinant chondroitinase AC; wherein M represents a molecular weight marker; the number 1 represents a bacterial liquid without an inducer, 2 represents a bacterial liquid induced after the inducer is added, 3 represents a precipitate after ultrasonic disruption, 4 represents a supernatant after the ultrasonic disruption, and 5 represents a purified target protein;
FIG. 3 is a graph showing the thermal stability analysis of recombinant chondroitinase AC at 37 ℃;
FIG. 4 is a graph showing the storage stability analysis of recombinant chondroitinase AC at 4 ℃.
Detailed Description
The invention discloses a recombinant chondroitinase AC and a preparation method and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. The methods, devices and materials used in the examples which follow, if not specifically indicated, are all conventional and commercially available methods, devices and materials used in the art.
Reagent:Pedobacter sp. ok626(GenBank: SDL 23104.1) was purchased from the Guangdong center for the preservation of microbial cultures; pET28a and a plasmid extraction kit are purchased from Biotechnology engineering (Shanghai) GmbH; peptone, yeast extract, glucose (PYG), chondroitin sulfate A, etc. were purchased from Sigma.
Example 1: cloning of chondrosulphatase AC gene
（1）Pedobacter sp. ok626Extraction of genomic DNA
1) To be activatedPedobacter sp. ok626The strain of (2) was inoculated into a 250 mL Erlenmeyer flask containing 100 mL peptone-yeast-glucose (PYG) medium in 10% CO2Then, the cells were cultured at 37 ℃ for 18 hours and centrifuged at 5866 Xg to obtain cells.
2) Washing the thalli centrifugally collected in the step 1) with distilled water with the volume 5 times that of the thalli, shaking, precipitating, mixing uniformly, centrifuging for 8 minutes at the temperature of 4 ℃ at 5866 Xg, and removing supernatant; repeating the steps once; adding absolute alcohol into the mortar, fully burning, and eliminating RNase possibly existing in the mortar; adding the thalli obtained by centrifugation into a mortar precooled by liquid nitrogen, and fully grinding;
3) plasmid extraction is carried out on the centrifuged thalli by using a plasmid extraction kit to obtainPedobacter spGenomic DNA of ok626, stored at-80 ℃.
(2) Gene for amplifying chondrosulphatase AC through PCR reaction
Found from NCBIPedobacter spThe chondrosulphatase AC gene of ok626, designing primers for amplifying the open reading frame of the chondrosulphatase AC gene, the designed substances respectively containingBamHI andXhoi the restriction sites of two enzymes: the sequence of the upstream primer is shown as SEQ ID NO: 1: 5'-acttatccttttggcaccataatgaaaagg-3', namely; the downstream primer is shown as seq id no: 2: namely 5'-ggttctagtaaagcagttcttttggctttt-3'.
As extracted in step (1)Pedobacter spOk626 genomic DNA as template, PCR amplification was performed using designed primers, reaction program: 1) 4 minutes at 95 ℃; 2) 30 seconds at 95 ℃; 3) 55 seconds at 60 ℃; 4) 1 minute at 72 ℃; 5) repeating steps 2) to 4) for 20 cycles; 5) at 72 ℃ for 10 minutes.
The PCR product was purified and used separately with expression vector pET28aBamHI andXhoi, double enzyme digestion is carried out, and the double enzyme digestion products are purified, recovered and then connected to obtain the recombinant expression vector pET-28 a-ChSaSeAC. The connection conditions are as follows: 12 hours at 25 ℃; ligation system (10 μ L): 2 mu L of vector, 6 mu L of target gene, 10 Xligation buffer1 mu L and 1 mu L of T4DNA ligase; and transforming the connected recombinant expression vector pET-28a-ChSaSeAC into an escherichia coli DH5 alpha competent cell, selecting a single colony for culturing, extracting a plasmid, and performing double enzyme digestion verification on the plasmid. FIG. 1 is a diagram of a double-restriction agarose electrophoresis analysis of a recombinant plasmid; wherein M represents a molecular weight marker; 1 is a recombinant expression vector pET-28 a-ChSaseAC; 2, 3 represents a double enzyme digestion product; such asAs shown in FIG. 1, the digestion results show that two bands correspond to pET28a (5369 bp) and chondroitinase sulfate AC (2040 bp), respectively, thereby verifying the successful connection of the recombinant expression plasmid.
After the colony containing the recombinant expression vector is subjected to amplification culture, a plasmid is extracted to store the glycerol strain, and the extracted plasmid is sent to the company of biological engineering (Shanghai) GmbH for sequencing. The total length of the gene of the chondroitin sulfate enzyme AC in the embodiment is 2097 bp through sequencing verification, and the nucleotide sequence is shown as SEQ ID NO. 3; the code is 701 amino acids, the molecular weight is 77kDa, and the amino acid sequence is shown in SEQ ID NO. 4. It should be noted that the chondroitinase AC of the present invention is not limited to the amino acid sequence shown in the sequence table of SEQ ID NO. 4, but may also be the amino acid sequence of a protein derived from the sequence shown in SEQ ID NO. 4 by substituting, deleting or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 4 and having the same protein activity.
Example 2: expression purification of chondrosulphatase AC
(1) The recombinant expression vector obtained in example 1 was transformed into E.coli BL21 (DE 3), and the strain was cultured. Then, a single colony was inoculated in LB liquid culture containing 5 mL of kanamycin (50. mu.g/mL) and cultured overnight at 37 ℃.
(2) 5 mL of overnight-cultured bacterial suspension was added to 250 mL of LB liquid medium (containing 50. mu.g mL)-1Kanamycin) in a 500 mL Erlenmeyer flask, and cultured at 200 rpm with shaking to OD600Approximately equal to 0.7.
(3) IPTG was added to a final concentration of 0.1mM and induced at 25 ℃ for 15 hours.
(4) Taking all culture solution, centrifuging at 5866 Xg for 5 minutes at 4 ℃; adding the centrifuged thallus into 8 mL Tris-HCl buffer (50 mM, pH 8.0), placing in ice water for ultrasonication, centrifuging at 4 deg.C for 20 min at 5866 Xg, and collecting supernatant to obtain crude enzyme solution of recombinant chondroitinase AC with total activity of 5082U L-1。
(5) And (3) adopting Ni column affinity purification to obtain the target protein. Draw 1 mL of Ni-NTA packing into the column, rinse with sterile water and equilibrate the Ni column with binding solution. The entire equilibrated filler was taken up and combined with the supernatant obtained in step (3) at 4 ℃ for 2 hours. The hetero-protein was washed with Tris-HCl buffer (pH 8.050 mM) containing 20 mM imidazole, eluted with Tris-HCl buffer (pH 8.050 mM) containing 250 mM imidazole, and the effluent was collected and confirmed by SDS-PAGE to be recombinant chondroitinase AC as the target protein.
FIG. 2 is an electrophoretic analysis chart of recombinant chondroitinase AC; wherein M represents a molecular weight marker; the number 1 represents a bacterial liquid without an inducer, 2 represents a bacterial liquid induced after the inducer is added, 3 represents a precipitate after ultrasonication, 4 represents a supernatant after ultrasonication, and 5 represents a purified target protein. As can be seen from FIG. 2, the purified target protein recombinant chondroitinase AC obtained a distinct band by SDS-PAGE, and the molecular weight was found to be 77kDa, which is the theoretical molecular weight.
Example 3: enzyme activity analysis of recombinant chondroitinase AC
In this example, the enzyme activity of chondroitinase AC was calculated by measuring the increase in absorbance at 232 nm of unsaturated disaccharide using chondroitin sulfate A as a substrate. The units of enzyme activity are: an amount of enzyme that degrades chondroitin sulfate A to form 1. mu. moL of unsaturated oligosaccharide product per minute at 37 ℃. The molar extinction coefficient of the product unsaturated oligosaccharide at 232 nm is 5100M−1cm−1And calculating the enzyme activity of the recombinant chondroitinase AC according to the protein standard curve. Each experiment was independently measured 3 times and the standard deviation calculated. The enzyme activity of the recombinant chondroitinase AC is calculated to be 325.2.6U mg-1-protein，K mThe value was 0.5mg mL-1，V maxIs 509.43U mg-1。
Example 4: stability analysis of recombinant chondroitinase AC
(1) Analysis of thermal stability
The recombinant chondroitinase sulfate AC obtained in example 2 was subjected to a thermal stability analysis at 37 ℃ without any enzyme protective agent.
Separately, 5. mu.L of purified recombinant chondroitinase AC was added to 895. mu.L of Tris-H pH 8.0Placing Cl buffer (50 mM) in 37 deg.C water bath for 5, 15, 30, 60, 120, 240, 360, 480, 600, 720 min, cooling at 4 deg.C for 1 min, and immediately adding 100 μ L10 mg/mL-1The reaction was started with the sodium chondroitin sulfate substrate solution and the enzymatic activity of the recombinant chondroitinase AC was measured. FIG. 3 is a graph showing the thermal stability analysis of recombinant chondroitinase AC at 37 ℃; as can be seen from FIG. 3, the recombinant chondroitinase AC can retain more than 90% of enzyme activity after being preserved at 37 ℃ for 60 min, and the half-life period at the temperature is 660 min. Through determination, the half-life period of the chondroitin sulfate enzyme AC derived from the Flavobacterium heparinum at 30 ℃ after being added with a maltose label is 498 min, and compared with the recombinant chondroitin sulfate enzyme AC provided by the invention, the recombinant chondroitin sulfate enzyme AC has good thermal stability.
(2) Storage stability analysis
10. mu.L of purified recombinant chondroitinase AC was pipetted into 1.5 mL EP tubes, centrifuged to ensure no air bubbles, stored in a refrigerator at 4 ℃ for 1, 2, 3, 4, 10, 15, 20, 25, and 30 days, and then taken out, and 890. mu.L of Tris-HCl buffer solution with pH 8.050 mM was added to measure the enzyme activity of the recombinant chondroitinase AC. FIG. 4 is a graph showing the storage stability analysis of recombinant chondroitinase AC at 4 ℃. As shown in figure 4, after the recombinant chondroitinase AC is stored for 4 days at 4 ℃, the enzyme activity loss is large and is 39%, the enzyme activity loss is stable and only decreases by 7% between 4 and 25 days, and the half-life period of the recombinant chondroitinase AC at 4 ℃ is 28 days, which indicates that the recombinant chondroitinase AC has good storage stability at 4 ℃ and good storage stability.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of Jiangsu
<120> recombinant chondroitinase AC, and preparation method and application thereof
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acttatcctt ttggcaccat aatgaaaagg 30
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ggttctagta aagcagttct tttggctttt 30
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cagtctggtg atgcggcggg tctgatcatg cagcgtatcg ttctggacct gcgtaaaccg 60
attcagtccg ttgataaagc agctgaaaag aacctggcaa ccctgcaggc taacggctct 120
tggaaaggca ttgattacac cgcaaaaacc atcgctaaat gggaaccggg cgatcacctg 180
gttaaactgg aaagcctggt gcaagcgtac gtgaccaaag actctcgtta cttctccaac 240
gatcaggttc tggaagctat cactctggct ctgaaacatt ggtacgatca ggacccgaaa 300
tccagcaact ggtggcataa cgaaatcgcg accccgcagg ctatcggtga actgctgatt 360
cagctgcgta acggcgaatc tcgtatcccg gcggatctgg aaagctccct gatcgaacgt 420
atgaaacgtg gtgatctggc taaacagacc ggtgcaaata aaactgacgt tgcgctgcac 480
tacttctacc gcgctctgct gaccaacaac caggcgctgc tggccacttc ttctgttgaa 540
ctgtttgatc cgatccgtct ggttcattat gatgaaggcc tgcagtatga ttattcttac 600
ctgcagcatg gtccgcagtt gcagatttct agctatggtg cggtatacat caccggcatc 660
ctgaaaatga tcaactacgt tcaaggtacc ccgtacgcga tcagcaacga aaaactgcag 720
ctgttctcta aatactaccg cgaaacctat ctgaaagcga tccgcggcag ctacatggat 780
ttcaacaccg aaggccgtgg cgtttctcgt ccgaacatcc tgcgtaaaaa cactgaaaaa 840
acccgcctga tcaccgctgt tctggttgat ccgctgcacg cggatgaatg gaaatccgct 900
atcgcgcgca ccgactcctc tgcggctcca gactacaaaa tcgaaccgct gcacaaacag 960
ttctggattg gcgactatgt gatgcacctg cgtccgggtt actccttcaa cgttcgcatg 1020
gtaagcaacc gtaccaaacg ctctgaatct ggcaacaaag aaaacctgtt tggtcgttat 1080
ctgtctgatg gcgcgactaa tatccaggtg cgtggcccgg aatattataa catcgtaccg 1140
atttgggaat gggacaaaat tccgggtacc accacccgtg attacgcaga agatcgtctg 1200
accaccaaat tctggggtga ggacggctct accgattttg taggtggcgt ttctgacggt 1260
gtttacggtg cttccaccta taccctgaac tatgatagcc tgtctggcaa aaaagcatgg 1320
ttcttctttg atcaagagat tgtgtgcctg ggcgctgata ttcgtagcaa caccccggaa 1380
agcattgtaa ccacggtgaa ccagtcttgg ctgaacggtg atgttcaggg ctctgctatt 1440
gaaggtaaat tcggcaaagg caaaaccgaa gtgttcaaaa acgaccacca gagttggatc 1500
ctgcacgacg gtattggtta cgttttcccg gaagcggcag atatcagcct gagcattaac 1560
acccagaaag gtagctggta caaaattaac aacgcaaaca gcaaaactga actgagcggc 1620
aacgttttta aactgtggat taaccatggc gctaaaccgg aagcagccaa atacgcatac 1680
gtggtgctgc cgggtatcaa aaacaccacc ccgctggtta actttggtaa atctggtatc 1740
cagatcctgg ctaatacccc gcaagtgcag gctgtttcca acgaacctct gaacatggtt 1800
caggccatct tttatgaacc gggcgtgctg aaagcgagcg cctacaccat taaatctgat 1860
aaagcgtgcg cactgctgat taaatctctg aacggtaaag tagtgattag cgtagcagat 1920
ccgaaacaga aagaacgtaa cgcggttatt agtatcacta acaacaaaga tggtaaaacc 1980
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Met Lys Arg Ile Leu Leu Ile Gly Val Leu Thr Leu Ser Met Leu Thr
1 5 10 15
Ser Arg Pro Ala Lys Ala Gln Ser Gly Asp Ala Ala Gly Leu Ile Met
20 25 30
Gln Arg Ile Val Leu Asp Leu Arg Lys Pro Ile Gln Ser Val Asp Lys
35 40 45
Ala Ala Glu Lys Asn Leu Ala Thr Leu Gln Ala Asn Gly Ser Trp Lys
50 55 60
Gly Ile Asp Tyr Thr Ala Lys Thr Ile Ala Lys Trp Glu Pro Gly Asp
65 70 75 80
His Leu Val Lys Leu Glu Ser Leu Val Gln Ala Tyr Val Thr Lys Asp
85 90 95
Ser Arg Tyr Phe Ser Asn Asp Gln Val Leu Glu Ala Ile Thr Leu Ala
100 105 110
Leu Lys His Trp Tyr Asp Gln Asp Pro Lys Ser Ser Asn Trp Trp His
115 120 125
Asn Glu Ile Ala Thr Pro Gln Ala Ile Gly Glu Leu Leu Ile Gln Leu
130 135 140
Arg Asn Gly Glu Ser Arg Ile Pro Ala Asp Leu Glu Ser Ser Leu Ile
145 150 155 160
Glu Arg Met Lys Arg Gly Asp Leu Ala Lys Gln Thr Gly Ala Asn Lys
165 170 175
Thr Asp Val Ala Leu His Tyr Phe Tyr Arg Ala Leu Leu Thr Asn Asn
180 185 190
Gln Ala Leu Leu Ala Thr Ser Ser Val Glu Leu Phe Asp Pro Ile Arg
195 200 205
Leu Val His Tyr Asp Glu Gly Leu Gln Tyr Asp Tyr Ser Tyr Leu Gln
210 215 220
His Gly Pro Gln Leu Gln Ile Ser Ser Tyr Gly Ala Val Tyr Ile Thr
225 230 235 240
Gly Ile Leu Lys Met Ile Asn Tyr Val Gln Gly Thr Pro Tyr Ala Ile
245 250 255
Ser Asn Glu Lys Leu Gln Leu Phe Ser Lys Tyr Tyr Arg Glu Thr Tyr
260 265 270
Leu Lys Ala Ile Arg Gly Ser Tyr Met Asp Phe Asn Thr Glu Gly Arg
275 280 285
Gly Val Ser Arg Pro Asn Ile Leu Arg Lys Asn Thr Glu Lys Thr Arg
290 295 300
Leu Ile Thr Ala Val Leu Val Asp Pro Leu His Ala Asp Glu Trp Lys
305 310 315 320
Ser Ala Ile Ala Arg Thr Asp Ser Ser Ala Ala Pro Asp Tyr Lys Ile
325 330 335
Glu Pro Leu His Lys Gln Phe Trp Ile Gly Asp Tyr Val Met His Leu
340 345 350
Arg Pro Gly Tyr Ser Phe Asn Val Arg Met Val Ser Asn Arg Thr Lys
355 360 365
Arg Ser Glu Ser Gly Asn Lys Glu Asn Leu Phe Gly Arg Tyr Leu Ser
370 375 380
Asp Gly Ala Thr Asn Ile Gln Val Arg Gly Pro Glu Tyr Tyr Asn Ile
385 390 395 400
Val Pro Ile Trp Glu Trp Asp Lys Ile Pro Gly Ile Thr Thr Arg Asp
405 410 415
Tyr Ala Glu Asp Arg Leu Thr Thr Lys Phe Trp Gly Glu Asp Gly Ser
420 425 430
Thr Asp Phe Val Gly Gly Val Ser Asp Gly Val Tyr Gly Ala Ser Thr
435 440 445
Tyr Thr Leu Asn Tyr Asp Ser Leu Ser Gly Lys Lys Ala Trp Phe Phe
450 455 460
Phe Asp Gln Glu Ile Val Cys Leu Gly Ala Asp Ile Arg Ser Asn Thr
465 470 475 480
Pro Glu Ser Ile Val Thr Thr Val Asn Gln Ser Trp Leu Asn Gly Asp
485 490 495
Val Gln Gly Ser Ala Ile Glu Gly Lys Phe Gly Lys Gly Lys Thr Glu
500 505 510
Val Phe Lys Asn Asp His Gln Ser Trp Ile Leu His Asp Gly Ile Gly
515 520 525
Tyr Val Phe Pro Ala Ala Asp Ile Ser Leu Ser Ile Asn Thr Gln Lys
530 535 540
Gly Ser Trp Tyr Lys Ile Asn Asn Ala Asn Ser Lys Thr Glu Leu Ser
545 550 555 560
Gly Asn Val Phe Lys Leu Trp Ile Asn His Gly Ala Lys Pro Glu Ala
565 570 575
Ala Lys Tyr Ala Tyr Val Val Leu Pro Gly Ile Lys Asn Ile Thr Pro
580 585 590
Leu Val Asn Phe Gly Lys Ser Gly Ile Gln Ile Leu Ala Asn Thr Pro
595 600 605
Gln Val Gln Ala Val Ser Asn Glu Pro Leu Asn Met Val Gln Ala Ile
610 615 620
Phe Tyr Glu Pro Gly Val Leu Lys Ala Ser Ala Tyr Thr Ile Lys Ser
625 630 635 640
Asp Lys Ala Cys Ala Leu Leu Ile Lys Ser Leu Asn Gly Lys Val Val
645 650 655
Ile Ser Val Ala Asp Pro Lys Gln Lys Glu Arg Asn Ala Val Ile Ser
660 665 670
Ile Thr Asn Asn Lys Asp Gly Lys Thr Thr Asp Tyr Pro Val Ser Phe
675 680 685
Pro Gln Gln Ala Phe Ala Gly Ala Thr Val Met Leu Lys
690 695 700
