CN113416725A - Recombinant chondroitinase ABC-I protein and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of bioengineering, and particularly relates to a recombinant chondroitinase ABC-I protein and a preparation method and application thereof. The invention uses molecular cloning technology to design primers by extracting chondroitinase ABC-I genes, 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, the activity of crude enzyme can reach 7144U/L, and the enzyme activity is 492.1U/mg after being purified by Ni-NTA; and the stability is good, the half-life period at 37 ℃ is 120 min, and the half-life period at 4 ℃ is 112 days. Has higher application value in the production of low molecular weight chondroitin sulfate and has wide prospect in the fields of medicine, environmental protection and the like.

Description

Recombinant chondroitinase ABC-I protein and preparation method and application thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a recombinant chondroitinase ABC-I protein and a preparation method and application thereof.

Background

Chondroitin sulfate (chondroitin sulfate) is a class of anionic biomacromolecules of glycosaminoglycans (GAGs), usually covalently linked to proteins. Plays an important role in processes including neuronal development, coagulation, inflammation, cell trafficking, cell proliferation and tumorigenesis. Chondroitin sulfate is a naturally-occurring component in connective tissues, and can be combined with water molecules to play a role in lubricating and supporting joints, so that the joints can move freely, and joint pain is relieved. In addition, it has also been shown to be useful as a food preservative with emulsifying properties and in nutritional formulations to prevent and reduce osteoarthritis. Chondroitin sulfate needs to function in cells, but the macromolecular volume of the chondroitin sulfate influences the smooth passing of the chondroitin sulfate through a layer of thin film on the cell surface, so that the chondroitin sulfate cannot play a plurality of good pharmacological functions in a body. The low molecular weight chondroitin sulfate has the advantages of good solubility, high bioavailability and the like, and has better biochemical characteristics.

Chondroitin sulfate ABC lyases (Chon-ABC, EC 4.2.2.4) are a class of glycosaminoglycans (GAGs) with broad specificity that degrade chondroitin sulfate glycosaminoglycans (GAGs) by β -elimination. According to the cleavage mechanism, Chon-ABCs are divided into endogenous (Chon-ABC-I, which depolymerizes chondroitin sulfate into unsaturated tetrasaccharides and disaccharides) and exogenous (Chon-ABC-II, which degrades chondroitin sulfate hexatetrasaccharides into disaccharides). The Chon-ABC-I has good medical prospect in the aspect of degrading chondroitin sulfate oligosaccharide. The chondroitinase ABC-I is mainly prepared by fermentation and purification processes, but has the problems of low enzyme activity, complex production, high cost and the like. In order to increase soluble expression and activity, chaperones were introduced for heterologous expression. But the production is still complicated, and the problems of inclusion body formation, low protein recovery rate and the like exist in the expression process. At present, a preparation process of chondroitinase ABC-I with low cost and high stability is not available.

Disclosure of Invention

In view of the above, the invention provides a recombinant chondroitinase ABC-I protein, a preparation method and an application thereof by a molecular cloning method. The recombinant chondroitinase ABC-I has higher enzyme activity and stability, simple preparation method and low cost, and can be used for industrial production of the chondroitinase ABC-I with low molecular weight.

The scheme of the invention is realized by the following technical means:

the invention provides a recombinant chondroitinase ABC-I protein, the nucleotide sequence of which is shown in SEQ ID NO. 3, the recombinant chondroitinase ABC-I protein encodes 1006 amino acids and has a molecular weight of 108 kDa.

On the other hand, the invention also provides an amino acid sequence for coding the chondroitinase ABC-I protein, and the amino acid sequence is shown as SEQ ID NO. 4.

The invention also provides a construction method of the recombinant chondroitinase ABC-I protein, which specifically comprises the following steps:

(1) carrying out PCR amplification coding by using a Bacteroides thetaiotaomicron chondroitinase ABC-I genome as a template and NdeI and XbaI enzyme cutting sites and 5' end protection bases as primers according to a conventional method to obtain a target gene segment;

(2) connecting the target gene fragment with an expression vector pCznl to construct a pCznl-Chon-ABC-I recombinant expression vector of the chondroitinase ABC-I gene;

(3) transforming the pCznl-Chon-ABC-I recombinant expression vector constructed in the step (2) into Escherichia coli host bacteria to construct chondrosulphatase ABC-I gene Escherichia coli engineering bacteria;

(4) induced expression of recombinant chondroitinase in chondroitinase ABC-I gene escherichia coli engineering bacteria;

(5) and (5) separating and purifying the expression product obtained in the step (4) to obtain the recombinant chondroitinase ABC-I 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 pCznl in the step (2) by using NdeI and XbaI 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 chondroitinase ABC-I gene escherichia coli engineering bacteria to 5 mL LB liquid culture medium containing 50 ug/mL kanamycin sulfate, performing shake culture at 37 ℃ overnight to obtain seed liquid, inoculating the seed liquid to fresh LB liquid culture medium, and performing shake culture at 37 ℃ until OD is achieved₆₀₀After reaching 0.6-0.8, IPTG was added to a final concentration of 0.75 mM and the mixture was shaken at 25 ℃ for 6 hours.

The molecular weight of the recombinant chondroitinase ABC-I protein in the step (5) is 108 kDa.

The invention also provides application of the recombinant chondroitinase ABC-I protein 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 ABC-I genes, 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, the activity of crude enzyme can reach 7144U/L, and the enzyme activity is 492.1U/mg after being purified by Ni-NTA; and the stability is good, the half-life period at 37 ℃ is 120 min, and the half-life period at 4 ℃ is 112 days. Has higher application value in the production of low molecular weight chondroitin sulfate and has wide prospect in the fields of medicine, environmental protection and the like.

Drawings

FIG. 1 is a diagram of a double-restriction agarose electrophoresis analysis of a recombinant plasmid; in the figure, A is PCR amplification product of ABC-I, and B is ABC-I recombinant plasmid;

FIG. 2 is an SDS-PAGE electrophoresis of recombinant chondroitinase ABC-I proteins;

FIG. 3 is a graph of the thermal stability analysis of recombinant chondroitinase ABC-I protein at 37 ℃;

FIG. 4 is a graph of a storage stability assay of recombinant chondroitinase ABC-I protein at 4 ℃.

Detailed Description

The invention discloses a recombinant chondroitinase ABC-I protein 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: bacteroides thetaiotaomicronBacteroides thetaiotaomicronPurchased in Guangdong microbial strain preservation center; pCznl and a plasmid extraction kit are purchased from Biotechnology engineering (Shanghai) GmbH; peptone, yeast extract, glucose (PYG), chondroitin sulfate A and the like were purchased from Sigma Co., Ltd.

Example 1: cloning of chondroitinase ABC-I gene

(1) Extraction of Bacteroides thetaiotaomicron genomic DNA

1) Activated Bacteroides thetaiotaomicronBacteroides thetaiotaomicron (PBD ID: 2Q1F) 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 1788 Xg to obtain cells.

2) Washing the centrifugally collected thalli with distilled water with 5 times of volume, shaking, precipitating, mixing uniformly, centrifuging for 8 minutes at 4 ℃ at 1788 Xg, and removing supernatant; this step was repeated once.

3) Adding anhydrous alcohol into the mortar, fully burning, and eliminating RNase possibly existing in the mortar.

4) And (3) adding the thalli obtained by centrifugation in the step 2) into a mortar precooled by liquid nitrogen, and fully grinding.

5) Plasmid extraction is carried out on the centrifuged thalli by using a plasmid extraction kit to obtainBacteroides thetaiotaomicronThe plasmid was stored at-80 ℃.

(2) Gene for amplifying chondroitinase ABC-I through PCR reaction

Found from NCBIBacteroides thetaiotaomicronThe gene of the chondroitinase ABC-I, a pair of primer sequences containing enzyme cutting sites are designed and synthesized for amplifying the open reading frame of the chondroitinase ABC-I gene, and the primers respectively contain the enzyme cutting sites of NdeI enzyme and XbaI enzyme: the sequence of the upstream primer is shown as SEQ ID NO: 1, and the following components: i.e. 5' -catatgcaggtcatagggtttgaag-3', and the sequence of the downstream primer is shown as seq id no: 2, as shown in the figure: i.e. 5' -agatcttttattttcaagaatcatttc-3’。

Bacteroides thetaiotaomicron as extracted in step (1)Bacteroides thetaiotaomicronUsing genome DNA as a template, and carrying out PCR amplification by using designed primers, wherein the reaction program comprises the following steps: 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. And (3) purifying the PCR product, performing double enzyme digestion on the PCR product and an expression vector pCznl by using NdeI and XbaI respectively, purifying and recovering the double enzyme digestion product, and connecting to obtain a recombinant expression vector pCznl-Chon-ABC-I. The connection condition is 25 ℃ and 12 hours; the ligation system (10. mu.L) was: 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.

And transforming the connected recombinant expression vector 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 vector; in the figure, A is a PCR amplification map of chondroitinase ABC-I, the number 1 indicates a DNA Marker, and the number 2 indicates a ChSase-ABC-I PCR amplification product; b is chondroitinase ABC-I recombinant plasmid; m is a DNA Marker, the number 1 represents ChSase-ABC-I recombinant plasmid, and the number 2 represents the restriction enzyme identification of the recombinant ChSase-ABC-I; as shown in FIG. 1, the digestion results show that the two target bands correspond to pCznl (5369 bp) and chondroitinase ABC-I (3018 bp), respectively, and that the recombinant expression plasmid is successfully ligated.

After the colony containing the recombinant expression vector pCznl-Chon-ABC-I is subjected to amplification culture, a plasmid is extracted to preserve the glycerol strain, and the extracted plasmid is sent to the company of bioengineering (Shanghai) GmbH for sequencing. The total length of the gene of the chondrosinase ABC-I sulfate in the embodiment is 3018 bp, the gene codes 1006 amino acids and has the molecular weight of 108 kDa through sequencing verification. The nucleotide sequence is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4. It should be noted that the chondroitinase ABC-I of the present invention is not limited to the amino acid sequence shown in the sequence table of SEQ ID NO. 4, but also can be the amino acid sequence of a protein which is 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 has the same protein activity.

Example 2: expression and purification of chondroitinase ABC-I

(1) The recombinant expression vector obtained in example 1 was transformed into E.coli BL21 (DE3), 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 transferred to a 500 mL Erlenmeyer flask containing 250 mL of LB liquid medium (containing 50. mu.g/mL kanamycin), and the mixture was shake-cultured at 200 rpm until OD₆₀₀0.6 to 0.8; adding isopropyl-beta-D-thioglycoside (IPTG) with the final concentration of 0.75 mM, and oscillating for 6 h at 25 ℃ to perform induced expression of the target gene;

(3) taking all culture solution, centrifuging at 1788 Xg for 5 minutes at 4 ℃; the centrifuged cells were added to 8 mL of PBS buffer (20 mM, pH 7.5, 200 mM NaCl, 10mM CaCl)₂) Placing the obtained product in ice water for ultrasonic disruption, centrifuging at 1788 Xg at 4 ℃ for 20 minutes, and collecting supernatant to obtain crude enzyme solution of recombinant chondroitinase ABC-I.

(4) And (3) adopting Ni column affinity purification to obtain the target protein. 1 mL of Ni-NTA filler was pipetted into a chromatographic column and washed with sterile waterWashing and balancing the Ni column with a binding solution; the entire equilibrated filler was taken up and combined with the supernatant obtained in step (3) at 4 ℃ for 2 hours. Washing the contaminating proteins with 20 mM PBS buffer containing 20 mM imidazole to OD of the effluent₂₈₀The value reached the baseline. Eluting with 20 mM PBS buffer containing 250 mM imidazole, collecting eluate, and verifying purified target protein recombinant chondroitinase ABC-I protein by SDS-PAGE.

FIG. 2 is an SDS-PAGE electrophoresis of recombinant chondroitinase ABC-I proteins; in the figure, M represents a molecular weight marker; the number 1 represents Marker, and the number 2 represents chondroitinase Chon-ABC-I; as shown in FIG. 2, recombinant chondroitinase ABC-I gave a clear band of interest at 108 kDa by SDS-PAGE, which is consistent with the theoretical value of chondroitinase ABC-I of 108 kDa.

Example 3: enzyme activity analysis of recombinant chondroitinase ABC-I protein

In this example, the activity of chondroitinase ABC-I was calculated by measuring the increase in absorbance at 232 nm of the unsaturated disaccharide using chondroitin sulfate A as a substrate. The units of enzyme activity are: an amount of an enzyme which degrades chondroitin sulfate A to form 1. mu. moL of unsaturated disaccharide per minute at 37 ℃. The molar extinction coefficient of the unsaturated disaccharide of the product at 232 nm is 3.8, and the enzyme activity of the chondroitinase ABC-I is calculated according to a protein standard curve. Each experiment was independently measured 3 times and the standard deviation calculated. The crude enzyme activity of the recombinant chondroitinase ABC-I protein can reach 7144U/L through calculation, and the enzyme activity is 492.1U/mg after Ni-NTA purification.

Example 4: analysis of thermal stability of recombinant chondroitinase ABC-I protein

(1) Analysis of thermal stability

The recombinant chondroitinase ABC-I protein obtained in example 2 was subjected to a thermostability assay at 37 ℃ without any enzyme protectant added during the assay.

Respectively placing 5 μ L purified recombinant chondroitinase ABC-I protein in 895 μ L PBS buffer solution (20 mM) with pH of 8.0, respectively placing in water bath at 37 deg.C for 0, 2, 10, 20, 30, 40, 60, 120 min, cooling at 4 deg.C for 1 min, immediately addingThe concentration of the mixed solution is 10 mg/mL in 100 mu L^-1The reaction is started and the enzyme activity of the recombinant chondroitinase ABC-I protein is measured. FIG. 3 is a graph of the thermal stability analysis of recombinant chondroitinase ABC-I protein at 37 ℃; as shown in figure 3, after the recombinant chondroitinase ABC-I protein is subjected to heat preservation at 37 ℃ for 60 min, more than 80% of enzyme activity can be reserved, and the half-life of the recombinant chondroitinase ABC-I protein at 37 ℃ is 120 minutes, so that the recombinant chondroitinase ABC-I protein has good thermal stability.

(2) Storage stability analysis

mu.L of the recombinant chondroitinase ABC-I protein purified in example 2 was pipetted into 1.5 mL EP tubes, centrifuged to ensure that no air bubbles were present, stored in a refrigerator at 4 ℃ for 0, 7, 10, 14, 21 and 35 days, and then taken out, and 890. mu.L of PBS buffer (20 mM) having a pH of 7.5 was added to determine the enzyme activity of the recombinant chondroitinase ABC-I protein. FIG. 4 is a graph of a storage stability assay of recombinant chondroitinase ABC-I protein at 4 ℃. As shown in figure 4, the recombinant chondroitinase ABC-I protein is stored in an environment of 4 ℃ with less activity loss and can be stored for a relatively long time, the recombinant chondroitinase ABC-I protein still has the enzyme activity of more than 80% on the 35 th day of storage, and the half-life period of the recombinant chondroitinase ABC-I protein is 112 days in the environment of 4 ℃, which indicates that the prepared recombinant chondroitinase ABC-I protein has 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

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gcaggatcac cggtccgcga ggaaatgaaa cggaaattcc ttgcgatgta cgaccatatc 1020

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agcattctga tgatggaaga cacgccggag aagctgcaat acttgaaatc cttctcccga 1320

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acgaatatga tttatctttt cagtcggaca agccttgccg tttccgaatt ggcacaccgg 1500

acggtcaaag atgtcctgct tgccatgcgt ttctactgca ataaactgaa ctttcccttg 1560

tctatgtccg ggcgccatcc cgatggacag gggaaattag ttcccatgca ctatgcgatg 1620

atggcaattg ccggaactcc cgatggaaag ggcgattttg ataaagagat ggcatccgcc 1680

tacctgcgtc tcgtatcttc cgattcctct tctgcggaac aggcaccgga atatatgccg 1740

aaagtttcga acgctcagga gcgtaagatt gccaaacgac tggtggaaaa cggttttcgt 1800

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cgtgaaaact ggtcggctgt ggcacgcgga cattcacgct acctttgggc agccgaacat 1920

tatctgggac acaacctcta tggacgttat ctggcacatg gaagtctcca gatactgaca 1980

gcacctccgg gacagacagt gactcccgcc accagcggat ggcagcagga aggtttcgac 2040

tggaaccgca ttccgggagt gacttccatc catctcccgc tggacctgct caaagccaac 2100

gtgctgaatg tagatacatt ctccggaatg gaagagatgc tttattcgga tgaagccttt 2160

gccggcggcc tgtcacaagg aaagatgaat ggcaacttcg gaatgaaact gcacgaacac 2220

gataagtata acggaacgca tcgtgcgcga aaatcctacc atttcatcga tggaatgatc 2280

gtttgtctag gctcggatat cgagaataca aacacggatt atccaacgga gacgaccatc 2340

ttccaactgg cggtgacaga taaagcggca catgactatt ggaagaataa tgcgggtgaa 2400

ggaaaagtat ggatggatca tctgggcacc ggatattatg tgccggttcc tgcaagattc 2460

gagaaaaact ttccgcaata ctcccgtatg caggatacag gcaaagaaac gaaaggagac 2520

tgggtatcat tgattatcga ccatgggaaa gcaccgaaag cgggaagcta cgaatatgcc 2580

attctgccgg gaaccgaccg gaagacgatg actgcctttg caaagaaacc tgcctacagc 2640

gtattacagc aagaccggaa tgcacatatc cttgaatcgc cttcggacag gataacttct 2700

tatgtgcttt ttgaaactcc ccagtcactg cttcccggtg gattgttgca gcgtacggat 2760

acgtcctgct tagtgatggt ccgtaaggaa tctgctgata aagtattatt gactgtagct 2820

caaccggatc tggcattgta tcgtggtccg agtgatgaag ctttcgataa agacggaaaa 2880

cgtatggaac gcagcatcta ttcccgcccg tggattgaca atgaaagcgg tgagattcct 2940

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Met Phe Ser Phe Glu Glu Pro Gln Leu Pro Ala Cys Ile Thr Gly Val

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Gln Ser Gln Leu Gly Ile Ser Gly Ala His Tyr Lys Asp Gly Lys His

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Tyr Phe Asp Leu Met Lys Arg Ile Ala Val Ala Tyr Asn Asn Ser Glu

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Ala Gly Ser Pro Val Arg Glu Glu Met Lys Arg Lys Phe Leu Ala Met

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Tyr Asp His Ile Thr Asp Gln Gly Val Ala Tyr Gly Ser Cys Trp Gly

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Asn Ile His His Tyr Gly Tyr Ser Val Arg Gly Leu Tyr Pro Ala Tyr

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Phe Leu Met Lys Asp Val Leu Arg Glu Glu Gly Lys Leu Leu Glu Ala

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Gly Thr Pro Asp Gly Lys Gly Asp Phe Asp Lys Glu Met Ala Ser Ala

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Tyr Leu Arg Leu Val Ser Ser Asp Ser Ser Ser Ala Glu Gln Ala Pro

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Glu Tyr Met Pro Lys Val Ser Asn Ala Gln Glu Arg Lys Ile Ala Lys

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Arg Leu Val Glu Asn Gly Phe Arg Ala Glu Ser Asp Pro Gln Gly Asn

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Leu Ser Leu Gly Tyr Gly Cys Val Ser Val Gln Arg Arg Glu Asn Trp

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Ser Ala Val Ala Arg Gly His Ser Arg Tyr Leu Trp Ala Ala Glu His

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Tyr Leu Gly His Asn Leu Tyr Gly Arg Tyr Leu Ala His Gly Ser Leu

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Gln Ile Leu Thr Ala Pro Pro Gly Gln Thr Val Thr Pro Ala Thr Ser

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Gly Trp Gln Gln Glu Gly Phe Asp Trp Asn Arg Ile Pro Gly Val Thr

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Ser Ile His Leu Pro Leu Asp Leu Leu Lys Ala Asn Val Leu Asn Val

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Asp Thr Phe Ser Gly Met Glu Glu Met Leu Tyr Ser Asp Glu Ala Phe

705 710 715 720

Ala Gly Gly Leu Ser Gln Gly Lys Met Asn Gly Asn Phe Gly Met Lys

725 730 735

Leu His Glu His Asp Lys Tyr Asn Gly Thr His Arg Ala Arg Lys Ser

740 745 750

Tyr His Phe Ile Asp Gly Met Ile Val Cys Leu Gly Ser Asp Ile Glu

755 760 765

Asn Thr Asn Thr Asp Tyr Pro Thr Glu Thr Thr Ile Phe Gln Leu Ala

770 775 780

Val Thr Asp Lys Ala Ala His Asp Tyr Trp Lys Asn Asn Ala Gly Glu

785 790 795 800

Gly Lys Val Trp Met Asp His Leu Gly Thr Gly Tyr Tyr Val Pro Val

805 810 815

Pro Ala Arg Phe Glu Lys Asn Phe Pro Gln Tyr Ser Arg Met Gln Asp

820 825 830

Thr Gly Lys Glu Thr Lys Gly Asp Trp Val Ser Leu Ile Ile Asp His

835 840 845

Gly Lys Ala Pro Lys Ala Gly Ser Tyr Glu Tyr Ala Ile Leu Pro Gly

850 855 860

Thr Asp Arg Lys Thr Met Thr Ala Phe Ala Lys Lys Pro Ala Tyr Ser

865 870 875 880

Val Leu Gln Gln Asp Arg Asn Ala His Ile Leu Glu Ser Pro Ser Asp

885 890 895

Arg Ile Thr Ser Tyr Val Leu Phe Glu Thr Pro Gln Ser Leu Leu Pro

900 905 910

Gly Gly Leu Leu Gln Arg Thr Asp Thr Ser Cys Leu Val Met Val Arg

915 920 925

Lys Glu Ser Ala Asp Lys Val Leu Leu Thr Val Ala Gln Pro Asp Leu

930 935 940

Ala Leu Tyr Arg Gly Pro Ser Asp Glu Ala Phe Asp Lys Asp Gly Lys

945 950 955 960

Arg Met Glu Arg Ser Ile Tyr Ser Arg Pro Trp Ile Asp Asn Glu Ser

965 970 975

Gly Glu Ile Pro Val Thr Val Thr Leu Lys Gly Arg Trp Lys Val Ala

980 985 990

Glu Thr Pro Phe Cys Lys Val Val Ser Glu Asp Lys Lys Gln Thr Val

995 1000 1005

Leu Arg Phe Leu Cys Lys Asp Gly Ala Ser Tyr Glu Val Glu Leu Glu

1010 1015 1020

Lys

1025

Claims (10)

1. The recombinant chondroitinase ABC-I protein is characterized in that the nucleotide sequence of the recombinant chondroitinase ABC-I protein is shown as SEQ ID NO. 3, 1006 amino acids are coded, and the molecular weight is 108 kDa.

2. The recombinant chondroitinase ABC-I protein as set forth in claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO 4.

3. A construction method of recombinant chondroitinase ABC-I protein is characterized by comprising the following steps:

(1) carrying out PCR amplification coding by using a Bacteroides thetaiotaomicron chondroitinase ABC-I genome as a template and NdeI and XbaI enzyme cutting sites and 5' end protection bases as primers according to a conventional method to obtain a target gene segment;

(2) connecting the target gene fragment with an expression vector pCznl to construct a pCznl-Chon-ABC-I recombinant expression vector of the chondroitinase ABC-I gene;

(3) transforming the pCznl-Chon-ABC-I recombinant expression vector constructed in the step (2) into Escherichia coli host bacteria to construct chondrosulphatase ABC-I gene Escherichia coli engineering bacteria;

(4) induced expression of recombinant chondroitinase in chondroitinase ABC-I gene escherichia coli engineering bacteria;

(5) and (5) separating and purifying the expression product obtained in the step (4) to obtain the recombinant chondroitinase ABC-I 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 according to claim 3, wherein the target gene fragment and the expression vector pCznl in step (2) are cleaved with NdeI and XbaI, respectively, in a double-restriction enzyme.

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 chondroitinase ABC-I gene escherichia coli engineering bacteria to 5 mL LB liquid culture medium containing 50 ug/mL kanamycin sulfate, performing shake culture at 37 ℃ overnight to obtain seed liquid, inoculating the seed liquid to fresh LB liquid culture medium, and performing shake culture at 37 ℃ until OD is achieved₆₀₀After reaching 0.6-0.8, IPTG was added to a final concentration of 0.75 mM and the mixture was shaken at 25 ℃ for 6 hours.

9. The method according to claim 3, wherein the molecular weight of the recombinant chondroitinase ABC-I protein of step (5) is 108 kDa.

10. Use of the recombinant chondroitinase ABC-I protein as claimed in claim 1 for the preparation of low molecular weight chondroitin sulfate.

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