CN105441471B - Chondroitin sulfate A (CSA) BC enzyme fusion proteins preparation method and applications - Google Patents
Chondroitin sulfate A (CSA) BC enzyme fusion proteins preparation method and applications Download PDFInfo
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Abstract
The present invention provides a kind of method of expression chondroitin sulfate A (CSA) BC enzyme fusion proteins, this method includes：1) chondroitin sulfate A (CSA) BC enzyme genes and 3 glyceraldehyde phosphate dehydrogenase protein gene are cloned respectively, and connect said gene so that the 3 glyceraldehyde phosphate dehydrogenase albumen subsequently expressed are connected with chondroitin sulfate A (CSA) BC zymoprotein N-terminals；2) by the chondroitin sulfate A (CSA) BC enzyme fusion proteins genetic recombination of connection to expression vector；3) expression vector containing chondroitin sulfate A (CSA) BC enzyme fusion proteins genes is transformed into host cell, is expressed；And 4) harvest and purify the chondroitin sulfate A (CSA) BC enzyme fusion proteins of expression.The activared carbon sulfur Chondroitin A BC enzyme fusion proteins obtained using this method account for 70% that summary table reaches protein content so that postorder purification process greatly simplifies.Obtained ChSase ABC I fusion proteins have high enzyme activity, can be used in producing chondroitin sulfate, and the formation condition of chondroitin sulfate is not high, method is easy and is easy to control.
Description
Technical field
The present invention relates to a kind of chondroitinase ABC fusion protein preparation methods in genetic engineering and field of fermentation engineering
And its application.
Background technology
Chondroitinase (chondroitinase or chondroitin sulfateyase, referred to as
" ChSase ") to be one kind can be degraded to the glycosaminoglycans such as chondroitin sulfate, chondroitin, hyaluronic acid unsaturated disaccharides (Δ Di
And oligosaccharides) lyases.
With the further investigation to ChSase, it is found that it is wide chondroitinase ABC (referred to as " ChSase ABC ") has
General application value.Scientific research personnel utilizes ChSase ABC detection chondroitin sulfates and production low-molecular weight chondroitin sulfate.Bao
The method of human relations army, the ChSase ABC enzymolysis-high performance liquid chromatography such as Yang Jiancheng detects the hyaluronic acid in shark's fin, uses
ZORBAX glycan analysis columns, ultraviolet detection wavelength are 226nm, detect that the mass fraction of hyaluronic acid in shark's fin is 0.86-
1.96% (enzymolysis of chondroitin sulfate-high effective liquid chromatography for measuring chromatographies, 2002,20 (6) in the shark's fins such as Bao Lunjun:
557-559)。
In addition ChSase ABC also have prodigious effect in terms of clinical application.In terms of alleviating retinosis, it can drop
Chondroitin sulfate proteoglycan (CSPGs) is solved, CSPGs is the important component of glial scar after central nervous system (CNS) damage,
It can inhibit neural axon regeneration.CSPGs mainly inhibits nerve regneration by sugared ammonia polysaccharide chain, therefore removes GAG or interfere it
Synthesis can make axon regeneration.Topaz seedling, Gao Pengfen etc. pass through intravitreal ChSase ABC enzymes, immunofluorescence technique observation
The expression of CSPGs, reverse transcription-polymerase chain reaction (RT-PCR) method detect the expression of versican mRNA, detection light
Receptor apoptosis situation, the results showed that Chase ABC can be denaturalized the CSPGs of rat retina abnormal deposition by degradation, inhibit
The apoptosis of photoreceptor cell, to promote to damage reparation (the chondrosulphatases such as the topaz seedling alleviation retinosis of nethike embrane
Rat photoreceptor cell apoptosis Yangzhou Universitys journal, 2012,33 (4):19-23.).
In terms of improving hind limb motor ability, ChSase ABC can repair spinal injury.Spinal cord injury is spine fracture
Common complication, quadriplegia can be caused, a series of severe complications such as gatism and breathing myoparalysis, after spinal cord injury
Nerve regneration and function reparation are a great problems in current medical domain.Sun Yongxin, Liu Ning etc. are combined using ChSase ABC
Hyperbaric oxygen preconditioning is to acetyl courage in different times hind limb motor function after spinal cord of adult rats damage and gastrocnemius motor end plate
The influence of alkali esterase (AChE) content, to detect the effect of ChSase ABC.The result shows that hyperbaric oxygen preconditioning can improve greatly
The motor function of mouse suffering limb and the activity for improving AChE, joint ChSase ABC act on stronger (the chondrosulphatases such as Sun Yongxin
ABC combines influence of the hyperbaric oxygen preconditioning to rats with spinal cord injury hind limb motor function and dissects scientific advance, 2012,18 (6):
526-529.).Nicole J.Tester etc. treat injured cat with ChSase ABC, and ChSase ABC have repaired the ridge of cat
Column damage, improve cat locomitivity (referring to：Nicole J.et al.Chondroitinase ABC improves
basic and skilled locomotion in spinal cord injured cats.Experimental
Neurology,2008,209(2):483-496)。
In recent years, Mark R Brown etc. it has been investigated that, key component in Lumbar intervertebral disc protrusion is proteoglycans, and is used
ChSase ABC and ChSase AC then can selective degradation fall intraspinal tube outstanding glycosaminoglycan, reduce intraspinal pressure, simultaneously
(Brown, M D.Method for treating intervertebral disc harmless to canalis spinalis peripheral tissues
displacement with enzymes[P]:US,4696816,1987-09-29).The discoveries such as Denholm EM in 2001
ChSase AC, ChSase B can inhibit the formation of melanoma blood vessel and the transfer of oncocyte and hyperplasia etc. (Denholm E M,
Lin Y Q,Silver P J.Anti-tumor activities of chondroitinase AC and
chondroitinase B:inhibition of angiogenesis,proliferation and
invasion.European Journal of Pharmacology,2001,416(3):213-221).The discoveries such as Lee MC are worked as
After cartilage ChSase ABC processing, the cartilage cell of transplanting and the Adhering capacity of the cartilage surface of a wound greatly enhance (Lee M C,
Sung K L,Kurtis M S,et a1.Adhesive force of chondrocytes to cartilage effects
of chondroitinase ABC.Clin Orthop,2000,370(1):286-294)。
To the preparation of low-molecular weight chondroitin sulfate frequently with acid-hydrolysis method, ion-exchange and enzymatic isolation method.First two side
Method needs more complex experimental facilities and pollutes the environment, comparatively, enzymatic isolation method need condition is high, method is easy and
It is easy to control.The key of enzymatic isolation method is to obtain a large amount of chondroitinase.Isolate and purify chondroitinase
Method it is extremely complex, it usually needs the chromatogram purification Jing Guo multistep, yield are very low.It is normal that heterologous recombination, which expresses ChSase ABC,
For improving one of the means of ChSase ABC yield.However it is very limited to the heterologous recombination expression study of ChSase ABC,
In the prior art use prokaryotic expression ChSase ABC zymoproteins after it is very low with ratio existing for activated protein state,
Albuminate need to pass through denaturation, renaturation process, and preparation process is cumbersome.
In addition, patent application CN103305496A discloses a kind of method for extracting chondrosulphatase from microorganism, but
It is that this method extraction step is cumbersome, cost is higher.
Invention content
To solve the deficiencies in the prior art, the present invention obtains great amount of soluble using a kind of different fusion tag methods
ChSase ABC I fusion protein, greatly reduce the complicated process of subsequent processing.
The main purpose of the present invention is to provide a kind of methods preparing chondroitin sulfate A (CSA) BC enzyme fusion proteins, wherein institute
The method of stating includes：1) it clones and connects chondroitin sulfate A (CSA) BC enzyme fusion proteins genes, clone the chondroitin sulfate A (CSA) BC respectively
Enzyme gene and glyceraldehyde 3-phosphate dehydro-genase protein gene, and connect said gene so that the glyceraldehyde 3-phosphate subsequently expressed
Apodehydrogenase is connected with chondroitin sulfate A (CSA) BC zymoprotein N-terminals；2) it obtains and contains the chondroitin sulfate A (CSA) BC enzyme fusion proteins
The recombinant expression carrier of gene carries the chondroitin sulfate A (CSA) BC enzyme fusion proteins genetic recombination of the step 1) connection to expression
Body；3) the chondroitin sulfate A (CSA) BC enzyme fusion proteins are expressed, contain chondroitin sulfate A (CSA) BC enzymes by what is cloned in the step 2)
The expression vector of antigen-4 fusion protein gene is transformed into host cell, is expressed；And 4) harvest and purify the sulphur of expression
Aching and limp ossein ABC enzyme fusion proteins.Preferably, chondroitin sulfate A (CSA) BC enzyme genes described in the step 1) encodes SEQ ID
NO:Amino acid sequence shown in 1；The glyceraldehyde 3-phosphate dehydro-genase gene code SEQ ID NO:Amino acid sequence shown in 2
Row.
Preferably, the chondroitin sulfate A (CSA) BC enzymes have SEQ ID NO:Amino acid shown in 1；The glycerol 3-phosphate
Aldehyde dehydrogenase albumen has SEQ ID NO:Amino acid shown in 2.
Preferably, chondroitin sulfate A (CSA) BC enzyme fusion proteins gene code SEQ ID NO in the step 1):Ammonia shown in 3
Base acid sequence.
Preferably, the fusion protein has SEQ ID NO:Amino acid shown in 3.
Preferably, the expression vector of expression chondroitin sulfate A (CSA) BC enzyme fusion proteins genes includes pMAL- in the step 2)
c2x。
Preferably, the host cell of the expression chondroitin sulfate A (CSA) BC enzyme fusion proteins includes in the step 3)
E.coliJM109 and DH5 α；Wherein, the expression chondroitin sulfate A (CSA) BC enzyme fusion proteins conditions are trained for TB in the step 3)
It supports 37 DEG C of base to cultivate 3 hours, final concentration of 0.5mM IPTG 10-42 DEG C Fiber differentiations is added 15-28 hours.
Preferably, the expression chondroitin sulfate A (CSA) BC enzyme fusion proteins conditions are 37 DEG C of TB culture mediums in the step 3)
Culture 3 hours is added 28 DEG C of final concentration of 0.5mM IPTG and induces 24 hours.
It is another aspect of the invention to provide chondroitin sulfate A (CSA) BC enzyme fusion proteins prepared by the method for the invention.
Preferably, the chondroitin sulfate A (CSA) BC enzyme fusion proteins enzyme activity is 30IU/mL, specific enzyme activity 37IU/mg.
It is another aspect of the invention to provide a kind of DNA, the DNA sequence encoding SEQ ID NO:Amino shown in 3
Acid sequence.
It is another aspect of the invention to provide a kind of recombinant vector, the recombinant vector includes carrier and of the present invention
DNA sequence dna, wherein the carrier includes pMAL-c2x.
It is another aspect of the invention to provide a kind of transformant, the transformant is by recombinant vector of the present invention
Import transformant obtained from host cell, wherein the host cell includes E.coli Top10, JM109, BL21, BL21
(DE3) and DH5 α.
It is described it is another aspect of the invention to provide a kind of method of production low-molecular weight chondroitin sulfate A, B or C
Method includes：1) it clones and connects chondroitin sulfate A (CSA) BC enzyme fusion proteins genes, clone the chondroitin sulfate A (CSA) BC enzymes respectively
Gene and glyceraldehyde 3-phosphate dehydro-genase protein gene, and connect said gene so that the glyceraldehyde 3-phosphate subsequently expressed is de-
Hydrogen zymoprotein is connected with chondroitin sulfate A (CSA) BC zymoprotein N-terminals；2) it obtains and contains the chondroitin sulfate A (CSA) BC enzyme fusion proteins bases
The recombinant expression carrier of cause, by the chondroitin sulfate A (CSA) BC enzyme fusion proteins genetic recombination of the step 1) connection to expression vector；
3) the chondroitin sulfate A (CSA) BC enzyme fusion proteins are expressed, are merged being cloned in the step 2) containing chondroitin sulfate A (CSA) BC enzymes
The expression vector of protein gene is transformed into host cell, is expressed；4) harvest and purify the chondroitin sulfate of expression
ABC enzyme fusion proteins；And 5) use the chondroitin sulfate A (CSA) BC enzyme fusion proteins degradation substrate of the step 4) purifying, reaction
Temperature is 30-55 DEG C, reaction time 5-10h, production low-molecular weight chondroitin sulfate A, B or C.
Preferably, chondroitin sulfate A (CSA) BC enzyme genes described in the step 1) encodes SEQ ID NO:Amino acid shown in 1
Sequence；The glyceraldehyde 3-phosphate dehydro-genase gene code SEQ ID NO:Amino acid sequence shown in 2.
Preferably, chondroitin sulfate A (CSA) BC enzyme fusion proteins gene code SEQ ID NO in the step 1):Ammonia shown in 3
Base acid sequence.
Preferably, the expression vector of expression chondroitin sulfate A (CSA) BC enzyme fusion proteins genes includes pMAL- in the step 2)
c2x。
Preferably, the host cell of the expression chondroitin sulfate A (CSA) BC enzyme fusion proteins includes in the step 3)
E.coliJM109 and DH5 α；Wherein, the expression chondroitin sulfate A (CSA) BC enzyme fusion proteins conditions are trained for TB in the step 3)
It supports 37 DEG C of base to cultivate 3 hours, final concentration of 0.5mM IPTG 10-42 DEG C Fiber differentiations is added 15-28 hours.
Preferably, the expression chondroitin sulfate A (CSA) BC enzyme fusion proteins conditions are 37 DEG C of TB culture mediums in the step 3)
Culture 3 hours is added 28 DEG C of final concentration of 0.5mM IPTG and induces 24 hours.
The present invention also provides the chondroitin sulfate A (CSA) BC enzyme fusion proteins to prepare low-molecular weight chondroitin sulfate A,
Application in B or C.
The low-molecular weight chondroitin sulfate A, B or the C that additionally provide the method and prepare of the present invention, wherein described
The molecular weight of chondroitin sulfate A (CSA), B or C are 0.5~25kDa, preferably ranging from 2~10kDa.
The present invention is merged using specific albumen with chondroitin sulfate A (CSA) BC enzymes, during recombinant expression, can be harvested and be accounted for expression
The activated protein of amount 70% enormously simplifies subsequent purifying procedure, expression product chondroitin sulfate A (CSA) BC enzyme fusion proteins, tool
The activity for having chondroitin sulfate A (CSA) BC enzymes, can be used in produce chondroitin sulfate, and the formation condition of chondroitin sulfate it is not high,
Method is easy and is easy to control.
Description of the drawings
Fig. 1 is the chondroitin sulfate A (CSA) BC enzyme gene electrophoresis patterns that PCR amplification obtains from proteus vulgaris, and 1-12 is each
Swimming lane corresponds to the PCR product expanded under the conditions of different annealing temperature respectively, and the corresponding annealing temperatures of swimming lane 1-12 are respectively 40.0
℃、40.6℃、41.9℃、44.0℃、46.4℃、48.7℃、51.3℃、53.6℃、56.0℃、58.1℃、59.4℃、60.0
℃。
Fig. 2 is transformant digestion verification electrophoretogram, and swimming lane 1 is the qualification result with Nde I and BamH I digestions, swimming lane 2
For with the qualification result of Nde I and Pst I digestions.
Fig. 3 is pMAL-c2x-ChSase ABC I and pMAL-c2x-gapA-ChSase ABC II different big at 6 kinds
Expression in enterobacteria (E.coli) host：Enzyme activity determination is using chondroitin sulfate A (CSA) and chondroitin sulfate B as substrate, wherein
Fig. 3 A are using chondroitin sulfate A (CSA) as host's optimum results of the pMAL-c2x-ChSase ABC I of substrate, and Fig. 3 B are soft with sulfuric acid
Ossein B is host's optimum results of the pMAL-c2x-ChSase ABC I of substrate, and Fig. 3 C are using chondroitin sulfate A (CSA) as substrate
Host's optimum results of pMAL-c2x-GAPDH-ChSase ABC I, Fig. 3 D are using chondroitin sulfate B as the pMAL- of substrate
Host's optimum results of c2x-GAPDH-ChSase ABC I.
Fig. 4 is E.coli JM109/pMAL-c2x-ChSase ABC I and E.coli JM109/pMAL-c2x-GAPDH-
The SDS-PAGE collection of illustrative plates of ChSase ABC I engineered strain expression products.
Fig. 5 is the SDS-PAGE collection of illustrative plates of ChSase ABC I and GAPDH-ChSase ABC I after purification.
In sequence table：
Sequence 1 is proteus vulgaris (Proteus vulgaris) chondroitin sulfate A (CSA) BC enzyme amino acid sequences；
Sequence 2 is Escherichia coli (Escherichia coli) glyceraldehyde 3-phosphate dehydro-genase protein amino acid sequence；
Sequence 3 is chondroitin sulfate A (CSA) BC enzyme fusion proteins amino acid sequences of the present invention；
Sequence 4 is chondroitinase ABC enzyme fusion proteins DNA sequence dna of the present invention；
Sequence 5 is the sense primer nucleotide sequence of PCR amplification chondroitin sulfate A (CSA) BC enzymes；
Sequence 6 is the downstream primer nucleotide sequence of PCR amplification chondroitin sulfate A (CSA) BC enzymes；
Sequence 7 is the sense primer nucleotide sequence of PCR amplification glyceraldehyde 3-phosphate dehydro-genase albumen；
Sequence 8 is the downstream primer nucleotide sequence of PCR amplification glyceraldehyde 3-phosphate dehydro-genase albumen.
Specific implementation mode
The invention will now be further described with reference to specific embodiments, and the advantages and features of the present invention will be with description more
It is clear.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
It should be understood that can be carried out without departing from the spirit and scope of the invention to the details and form of technical solution of the present invention
Modifications or substitutions, but these modifications and replacement are each fallen in protection scope of the present invention.
Hereinafter, embodiments described herein is specifically described.
Chondroitin sulfate A (CSA) BC enzyme fusion proteins
Present document relates to chondroitin sulfate A (CSA) BC enzyme fusion proteins (hereinafter, being also referred to as " GAPDH-ChSase ABC sometimes
I "), fusion has chondroitin sulfate A (CSA) BC enzymes and glyceraldehyde 3-phosphate dehydro-genase albumen (hereinafter, being also referred to as sometimes
“GAPDH”)。
Present document relates to chondroitin sulfate A (CSA) BC enzymes can be any chondrosulphatase, be preferably selected from proteus vulgaris
(Proteus vulgaris), Aeromonas hydrophila (Aeromonas liquefaciens) and Flavobacterium heparinum
The chondrosulphatase in the source (Flavobacterium heparinum).According to this paper preferred embodiment, sulphur
Aching and limp ossein ABC enzymes are the chondrosulphatases in proteus vulgaris source；Particularly, chondrosulphatase is to eliminate coding
The chondroitin sulfate A (CSA) BC enzymes of the proteus vulgaris of signal peptide sequence.According to a preferred embodiment herein, this paper sulfuric acid
Chondroitin A BC enzymes have SEQ ID NO:Amino acid shown in 1.
In chondroitin sulfate A (CSA) BC enzyme fusion proteins, chondroitin sulfate A (CSA) BC enzymes directly with glyceraldehyde 3-phosphate dehydro-genase albumen
Fusion.
Present document relates to glyceraldehyde 3-phosphate dehydro-genase albumen can be any those of ordinary skill in the art can obtain
Glyceraldehyde 3-phosphate dehydro-genase albumen.Come preferably from the glyceraldehyde 3-phosphate dehydro-genase albumen of Escherichia coli.More at one
In preferred embodiment, the glyceraldehyde 3-phosphate dehydro-genase albumen of this paper has SEQ ID NO:Amino acid shown in 2.
It is preferred that the chondroitin sulfate A (CSA) BC enzyme fusion proteins of this paper have SEQ ID NO:Amino acid shown in 3.
DNA
A kind of DNA of the above-mentioned all chondroitin sulfate A (CSA) BC enzyme fusion proteins of coding is also related to, is the fusion protein
Coded sequence.
Term " coded sequence " for this specification means the amino acid sequence for directly specifying its protein product
Nucleotide sequence.
The boundary of coded sequence is usually determined by opening frame, described to open frame usually with ATG initiation codon or for choosing
The initiation codon selected such as GTG and TTG start, and are terminated with terminator codon such as TAA, TAG and TGA.Coded sequence
Can be DNA, cDNA or recombinant nucleotide sequence.
According to a preferred embodiment herein, DNA has SEQ ID NO:Base sequence shown in 4.
Recombinant vector
Recombinant vector is also related to, it includes encode above-mentioned chondroitin sulfate A (CSA) BC enzyme fusion proteins DNA.
Present document relates to recombinant vector include the DNA sequence dna for encoding above-mentioned all fusion proteins, promoter, transcribe and turn over
Translate termination signal.DNA sequence dna described herein can be combined together with other regulating and controlling sequences, generate recombinant vector, the load
Body may include one or more (several) convenient restriction site to allow to be inserted into or replace coding peptide fragment in these sites
DNA sequence dna.Alternative, it can be by being inserted into the DNA sequences for including the amino acid sequence in the carrier appropriate for expressing
It arranges to express the DNA sequence dna of this paper.During Prepare restructuring carrier, coded sequence is imported in carrier, thus by the volume
Code sequence is operably connected with expression regulation sequence appropriate.
Promoter, transcription signal, translation termination signal and other regulating and controlling sequences are any those of ordinary skill in the art
Can according to conventional selection and determination.
Recombinant vector can be any carrier (for example, plasmid or virus), can easily carry out recombinant DNA step,
And the expression of nucleotide sequence can be generated.It is thin with the host that will introduce the carrier that the selection of carrier will generally depend on carrier
The compatibility of born of the same parents.Carrier can be linear or closed hoop plasmid.
Carrier can be autonomously replicationg vector, for example, plasmid, extra-chromosomal element, minichromosome or artificial chromosome.
Carrier can contain any structure for ensuring self-replacation.It ought be introduced into host cell alternatively, carrier can be one kind
When, the carrier that is integrated into genome and is replicated together with the chromosome for incorporating the carrier.In addition it is possible to use individually
Carrier or plasmid or two or more carriers or plasmid contain the global DNA of host cell gene group to be introduced jointly, or
Transposons can be used.
According to a preferred embodiment herein, the recombinant vector of this paper is pMAL-c2x.It is preferred that the recombinant vector is
It is built by following steps：
By SEQ ID NO:DNA fragmentation shown in 4 is inserted into the restriction enzyme site region of plasmid pMAL-c2x, obtains recombination and carries
Body.
Transformant
Transformant is also related to, it will includes to encode above-mentioned chondroitin sulfate A (CSA) BC enzyme fusion proteins DNA to import host to be
Transformant obtained from cell.
Present document relates to transformant can be used for the production of protein, by the vector introduction place that will include this paper DNA sequence dnas
Chief cell and obtain the transformant, and cultivate the transformant in culture medium appropriate, can be used for producing required target
Protein.
Term " host cell " includes the spawn of mother cell, different due to the mutation occurred in reproduction process
In mother cell.Host cell can be useful any cell in the recombination of the peptide fragment of this paper generates, for example, protokaryon or true
Nucleus.Prokaryotic host cell can be any gram-positive bacterium or gramnegative bacterium.Gram-positive bacterium packet
It includes but is not limited to, bacillus, streptococcus, streptomyces, staphylococcus, enterococcus spp, genus lactubacillus, galactococcus
Category, fusobacterium, ground bacillus category and bacillus marinus category.Gramnegative bacterium includes but not limited to Escherichia coli, vacation
Zygosaccharomyces, Salmonella, campylobacter, Helicobacterium, Flavobacterium, Fusobacterium, mud Bacillus, eisseria
And Ureaplasma.Bacterial host cell can be any bacillus cell.The useful bacillus in the implementation of this paper
It includes but not limited to Alkaliphilic bacillus, bacillus amyloliquefaciens, bacillus brevis, Bacillus circulans, gram Lloyd's to belong to cell
It is bacillus, bacillus coagulans, bacillus firmus, bacillus lautus, bacillus lentus, bacillus licheniformis, huge
Bacterium anthracoides, bacillus pumilus, bacillus stearothermophilus, bacillus subtilis and Bacillus thuringiensis cell.Carefully
Bacterium host cell can also be any streptococcus cell.Useful streptococcus cell includes but unlimited in the implementation of this paper
In streptococcus equisimilis, streptococcus pyogenes, streptococcus uberis and streptococcus equi subsp blast cells.Bacterial host cell can be with
It is any Streptomyces cell.Useful Streptomyces cell includes but not limited in the implementation of this paper, not streptomyces chromogenes,
Deinsectization streptomycete, streptomyces coelicolor, streptomyces griseus and shallow Streptomyces glaucoviolaceus cell.Host cell can also be eucaryote,
Such as mammal, insect, plant or fungal cell.In a specific aspect, host cell is fungal cell.It is specific at one
Aspect, fungal host cells are yeast cells.At another specific aspect, fungal host cells are filamentous fungal cells.
According to a preferred embodiment herein, bacterial host cell is Escherichia coli.According to further preferred reality
Scheme is applied, bacterial host cell is e. coli jm109.
The method for building chondroitin sulfate A (CSA) BC enzyme fusion proteins
A kind of method of structure chondroitin sulfate A (CSA) BC enzyme fusion proteins is also related to, is by glyceraldehyde 3-phosphate dehydrogenation
Enzyme gene and chondroitin sulfate A (CSA) BC enzyme genes are connected to pMAL-c2x simultaneously.
Another embodiment of this paper is related to the method for building chondroitin sulfate A (CSA) BC enzyme fusion proteins, wherein the sulfuric acid is soft
The N-terminal of ossein ABC enzymes passes through peptide fragment coupling part and glyceraldehyde 3-phosphate dehydro-genase protein binding.
The plasmid vector being directed to is as described above.
Recombinant vector is imported into host cell and obtains transformant；And
Using the medium culture transformant and obtain the crude products of the sulfur acid Chondroitin A BC enzyme fusion proteins.
Wherein, the culture of transformant can use the conventional nutrient for including carbon source, nitrogen source, inorganic salts, various vitamins etc.
Culture medium carries out, and as carbon source, can use such as glucose, sucrose, fructose, maltose carbohydrate, the alcohol such as ethyl alcohol, methanol
Class, the organic acids such as citric acid, malic acid, succinic acid, maleic acid, fumaric acid, blackstrap etc..As nitrogen source, can individually or
It is used in mixed way such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea.In addition, as inorganic salts, such as phosphoric acid can be used
One hydrogen potassium, potassium dihydrogen phosphate, magnesium sulfate etc..In addition, peptone, meat extract, yeast extract, corn can be added in culture medium
The nutrients such as the various vitamins such as slurry, casamino acid, biotin.
Culture usually carries out under the aerobic conditions such as air agitation, oscillation.Cultivation temperature is not particularly limited, as long as
Host microorganism is capable of the temperature of growth reproduction, in addition, being also not particularly limited to the pH in incubation, as long as place
Main microorganism is capable of the pH of growth reproduction.PH adjustment in culture can be carried out by adding acid or alkali.
It is the training carried out under conditions of induction to above-mentioned transformant culture according to a preferred embodiment herein
It supports, thus expression obtains chondroitin sulfate A (CSA) BC enzymes.The condition of Fiber differentiation is：The IPTG of 0.5mM.Further preferably at one
Embodiment in, above-mentioned inducing culturing condition is：10-42 DEG C of Fiber differentiation 15-28 hours.In terms of one is preferred,
Above-mentioned inducing culturing condition is：28 DEG C of Fiber differentiations 24 hours.
According to a preferred embodiment herein, the solvent for carrying out the culture medium of above-mentioned Fiber differentiation is water, and solute is
12.54g/L K2HPO4, 2.31g/L KH2PO4, 24g/L yeast extracts, 12g/L peptones, 4ml/L glycerine and 100 μ g/L
Ampicillin.
Method for the crude product for preparing sulfur acid Chondroitin A BC enzyme fusion proteins, is not particularly limited, can make
With various methods.In general, can prepare as follows：Using the transformant of above-mentioned medium culture appropriate, by using super
It is broken that the chemical methodes such as surfactant or enzymatic treatment etc. is either utilized in the physical methods such as sound wave, squeezing, osmotic shock
It is broken or solubilized, and the operations such as centrifugation or filtering, to obtain the thick production of sulfur acid Chondroitin A BC enzyme fusion proteins
Object.
According to a preferred embodiment herein, by the transformant culture thalline of above-mentioned culture using ultrasonic wave, pressure
The physical methods such as squeezing, osmotic shock, which be crushed, obtains the crude product of sulfur acid Chondroitin A BC enzyme fusion proteins.
According to a preferred embodiment herein, sulfur acid Chondroitin A BC enzyme fusion proteins are obtained using ultrasonication
Crude product.
To be the crude product of above-mentioned sulfur acid Chondroitin A BC enzyme fusion proteins according to a preferred embodiment herein
The affine separation of a step is realized by Ni columns.The Ni columns are by pre-equilibration.
The production method of low-molecular weight chondroitin sulfate A or C
Present document relates to the methods of production low-molecular weight chondroitin sulfate A, B or C comprising：Use chondroitin sulfate A (CSA) BC
Enzyme fusion proteins are degraded chondroitin sulfate A (CSA), the method for B either C raw materials, production low-molecular weight chondroitin sulfate A B or C,
In the obtained molecular weight of low-molecular weight chondroitin sulfate A, B or C be 0.5kDa~25kDa, preferably ranging from 2~
10kDa。
When the molecular weight of chondroitin sulfate is reduced to 2kDa~10kDa, drug effect becomes apparent, to preventing artery congee
Sample hardening, rheumatic inflammation and wound healing have more preferable curative effect.
Substrate for producing chondroitin sulfate can be selected arbitrarily, if this can be degraded by chondroitin sulfate A (CSA) BC enzymes
Substrate.It can be selected from：It is any in chondroitin sulfate A (CSA), chondroitin sulfate B, chondroitin sulfate C and hyaluronic acid.
According to this paper preferred technical solution, chondroitin sulfate is selected from chondroitin sulfate A (CSA), chondroitin sulfate B or chondroitin sulfate
Plain C.
The concentration for producing the substrate of chondroitin sulfate is that those skilled in the art can be according to the chondroitin sulfate added
The concentration of ABC enzyme fusion proteins and be suitably determined.The time that chondroitin sulfate is reacted with chondroitin sulfate A (CSA) BC enzyme fusion proteins
It can also be determined according to the molecular weight of the low-molecular weight chondroitin sulfate of target.
Embodiment
With reference to specific embodiment to being described further herein, but it is not limited to following embodiment herein.
It is conventional method unless otherwise specified in following embodiments.The primer synthesis and examining order are by Hua Da
Gene is completed, Q5TM2 × Master of High-Fidelity Mix and all restriction enzymes are purchased from New England
Biolabs companies；All competent cells are (such as：DH5 α, JM109 and Top10) it is purchased from the limited public affairs of Beijing Bo Maide biotechnologys
Department)；Chondroitin sulfate A (CSA) BC enzyme enzyme activity determination substrate chondroitin sulfate A (CSA)s are purchased from Nanjing Olympic Duo Fu Buddhist nun bio tech ltd,
Its chemicals is analytical reagents, is purchased from Beijing Chemical Plant.
The expression of embodiment 1, chondroitinase ABC fusion protein (GAPDH-ChSase ABC I)
1. removing the proteus vulgaris chondroitin sulfate A (CSA) BC enzymes coded sequence and glyceraldehyde 3-phosphate dehydro-genase of signal peptide
The clone of DNA sequence dna
The detailed process of expression vector pMAL-c2x-ChSase ABC I structures is as follows：
The design and synthesis of 1.1 primers
It inquires to obtain the DNA of proteus vulgaris chondroitin sulfate A (CSA) BC enzymes and glyceraldehyde 3-phosphate dehydro-genase through Genbank
Sequence, upstream and downstream primer used are respectively：
Sense primer P1：5’-CGGGATCCATGGCCACCAGCAATCCTGCATT-3’(SEQ ID NO:5) (with lower stroke
The base of line is the restriction enzyme site of BamH I),
Downstream primer P2：5’-AACTGCAGTTATCAAGGGAGTGGCGAGAGTTTG-3’(SEQ ID NO:6) (under band
The base of scribing line is the restriction enzyme site of Pst I), after amplification, i.e., BamH I and Pst I restriction enzyme sites are introduced respectively.
Sense primer P1：5’-GGAATTCCATATGACTATCAAAGTAGGTATCAACGGTTTTGGC-3’(SEQ ID
NO:7) (base with underscore is the restriction enzyme site of Nde I),
Downstream primer P2：5’-CGCGGATCCTTTGGAGATGTGAGCGATCAGGTC-3’(SEQ ID NO:8) (under band
The base of scribing line is the restriction enzyme site of BamH I), after amplification, i.e., Nde I and BamH I restriction enzyme sites are introduced respectively.
1.2 PCR amplifications remove proteus vulgaris chondroitin sulfate A (CSA) BC enzymes and the glyceraldehyde 3-phosphate dehydrogenation of signal peptide
The coded sequence of enzyme
The chondroitin sulfate A (CSA) BC enzyme reaction systems of PCR amplification are：50ng proteus vulgaris genomic DNA masterplates,
Each primer of 100pmol responds 2 × Master Mix of volume；Amplification program is：98 DEG C of pre-degenerations 30 seconds, 98 DEG C of denaturation 7
Second, 40-60 DEG C of primer annealing 30 seconds, 72 DEG C extend 2 minutes, and after 30 cycles, 72 DEG C extend that reaction was completed 2 minutes.The PCR is tied
Fruit is as shown in Figure 1, show that amplification has obtained the chondroitinase ABC genetic fragment of 3kb, sequencing shows and compares correct.Fig. 1
In, swimming lane 1-12 is respectively that annealing temperature is 40-60 DEG C of amplification, and swimming lane M is that (stripe size is followed successively by molecular weight marker
10kb, 8kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1kb), it is 3kb target fragments at arrow meaning, from left to right each swimming lanes of 1-12
Corresponding annealing temperature is respectively 40.0 DEG C, 40.6 DEG C, 41.9 DEG C, 44.0 DEG C, 46.4 DEG C, 48.7 DEG C, 51.3 DEG C, 53.6 DEG C,
56.0℃、58.1℃、59.4℃、60.0℃.As shown in Figure 1, optimal annealing temperature is 60 DEG C, is produced for PCR at arrow instruction
Object band.
For example above-mentioned 1.2PCR of the glyceraldehyde 3-phosphate dehydro-genase reaction system and method for PCR amplification expands chondroitin sulfate
ABC enzyme reactions system and method.
1.3 cloning vectors of the structure containing target fragment
The chondroitin sulfate A (CSA) BC enzyme dna sequences of BamH I and Pst I digestion PCR amplifications are used respectively, while using Nde
The glyceraldehyde 3-phosphate dehydro-genase DNA sequence dna of I and BamH I digestion PCR amplifications detaches digestion products, by digestion treated sulphur
Aching and limp ossein ABC enzyme dnas sequence and glyceraldehyde 3-phosphate dehydro-genase DNA sequence dna and the pMAL-c2x after Nde I and Pst I digestions
Carrier is connected with ligase, obtains recombinant vector.
The screening and sequencing of 1.4 conversion Escherichia coli and positive colony transformant
The recombinant vector that step 1.3 is obtained converts bacillus coli DH 5 alpha competent cell, and specific method is：By 10 μ l's
The bacillus coli DH 5 alpha competent cell mixing of connection product and 50 μ l, ice bath 30 minutes, 42 DEG C of heat shocks 90 seconds, ice bath 2 minutes,
Then it is added in 500 μ l LB liquid mediums (peptone 3g, yeast extract 1.5g, NaCl 3g, water 300mL), 37 DEG C incubate
Educate 60 minutes, be applied to containing 100 μ g/mL ampicillins LB resistance cultures tablet (peptone 3g, yeast extract 1.5g,
NaCl 3g, agar powder 4.5g, water 300mL) it is screened.37 DEG C are cultivated 12-20 hours.
Choosing colony and as template, bacterium colony PCR identifications, PCR reaction systems and reaction item are carried out with primer P1 and P2
Part is identical as step 1.2.
Digestion verification, such as Fig. 2, swimming lane 1,2 are the different bands that positive transformant is removed with different digestions, and swimming lane 1 is to use
The qualification result of Nde I and BamH I digestions, swimming lane 2 are the qualification result with Nde I and Pst I digestions, target item occur
Band, swimming lane M are molecular weight marker.
The positive colony that screening obtains is gone in LB liquid mediums of the 4mL containing 100 μ g/mL ampicillins, 37 DEG C,
220rpm shakes 12 hours, transfers to Hua Da gene to be sequenced bacterium solution, will contain chondroitin sulfate A (CSA) BC zymoprotein encoding genes
PMAL-c2x recombinant vectors be named as pMAL-c2x-GAPDH-ChSase ABC I.
By sequencing, it is corresponding to derive it as shown in SEQNO.1 for the gene order of fusion protein GAPDH-ChSase ABC I
Fusion protein amino acid sequence such as sequence table SEQ ID No:Shown in 3；In fusion protein GAPDH-ChSase ABC I
GAPDH amino acid sequence such as SEQ ID No:Shown in 2；The amino acid sequence of ChSase ABC I in the fusion protein is such as
SEQ ID No:Shown in 1.
2. the expression of chondroitin sulfate A (CSA) BC enzyme fusion proteins GAPDH-ChSase ABC I
The plasmid in bacillus coli DH 5 alpha containing pMAL-c2x-ChSase ABC I in extraction step 1.4, according to routine
Method converts Escherichia coli Top10, JM109, BL21 and BL21 (DE3)).
It is screened by ampicillin and carries out bacterium colony PCR identifications using the primer that step 1.1 provides, contained
Bacillus coli DH 5 alpha, Top10, JM109, BL21 and BL21 (DE3) of pMAL-c2x-GAPDH-ChSase ABC I, i.e.,
Top10/pMAL-c2x-GAPDH-ChSase ABC I、JM109/pMAL-c2x-GAPDH-ChSase ABC I、BL21/
PMAL-c2x-GAPDH-ChSase ABC I, BL21 (DE3)/pMAL-c2x-GAPDH-ChSase ABC I and DH5 α/pMAL-
Engineering bacterias of the c2x-GAPDH-ChSase ABC I as expression pMAL-c2x-GAPDH-ChSase ABC I.
With plasmid pMAL-c2x conversion Escherichia coli Top10, JM109, BL21, BL21 (DE3) and DH5 α, empty carrier is obtained
Control Top10/pMAL-c2x, JM109/pMAL-c2x, BL21/pMAL-c2x, BL21 (DE3)/pMAL-c2x and DH5 α/
pMAL-c2x。
It is operated below to the parallel progress of engineering bacteria above.
By empty vector control and engineering bacteria respectively in TB culture mediums (the 12.54g/L K containing amicillin resistance2HPO4,
2.31g/L KH2PO4, 24g/L yeast extracts, 12g/L peptones, 4ml/L glycerine contains 100 μ g/L ampicillins) and 37 DEG C
After culture 3 hours, 28 DEG C of final concentration of 0.5mM IPTG are added and induce 24 hours.
6000rpm, thalline were collected by centrifugation within 6 minutes and is washed twice with 20mM Tris-HCl (pH 7.4), and bacterium is then resuspended
Body.Re-suspension liquid is carried out ultrasonication, and (output power 300W, ultrasound 5 seconds and intermittently 6 seconds, processing total time are 15 points every time
Clock), 6000rpm is centrifuged for 6 minutes, and the supernatant of centrifugation gained is the thick production of sulfur acid Chondroitin A BC enzymes after ultrasonication
Object.
The detection of enzyme activity (unit IU/L) uses the optical absorption method of 232nm, the enzyme activity of 1 IU to be defined as 37 DEG C every point
Clock generates the reaction effect of 1 μm of oL unsaturated bond.Take chondroitin sulfate A (CSA) substrate solution 2mL (20mg/mL chondroitin sulfate A (CSA)s,
20mM Tris-HCl, pH 7.4), the crude product of 200 μ L of gained in walking in addition, final reaction volume is 1.5mL, is surveyed
In the absorbance change △ A of 232nm in unit interval232.Extinction coefficient epsilon=3800M-1。
The definition of specific enzyme activity (unit is IU/mg albumen) is the ratio of enzyme activity and crude product albumen concentration (unit mg/L)
Value.Albumen concentration monitor is using conventional Bradford methods.
Using chondroitin sulfate A (CSA) and B as host's optimum results of substrate as shown in figure 3, Fig. 3 A and Fig. 3 B are pMAL-c2x-
Host's optimum results of ChSase ABC I, Fig. 3 A are using chondroitin sulfate A (CSA) as the pMAL-c2x-ChSase ABC I's of substrate
Host's optimum results, Fig. 3 B are using chondroitin sulfate B as host's optimum results of the pMAL-c2x-ChSase ABC I of substrate；
Fig. 3 C and Fig. 3 D are host's optimum results of pMAL-c2x-GAPDH-ChSase ABC I, and Fig. 3 C are using chondroitin sulfate A (CSA) the bottom of as
Host's optimum results of the pMAL-c2x-GAPDH-ChSase ABC I of object, Fig. 3 D are using chondroitin sulfate B as substrate
Host's optimum results of pMAL-c2x-GAPDH-ChSase ABC I, it is visible for pMAL-c2x-GAPDH- by Fig. 3 C, 3D
For ChSase ABC I expression vectors, using Top10, BL21 and BL21 (DE3) as host expresses its supernatants in do not produce
Liveliness proof albumen (does not have enzyme activity, the longitudinal axis zero).
Empty vector control bacterial strain Top10/pMAL-c2x, JM109/pMAL-c2x, BL21/pMAL-c2x, BL21 (DE3)/
Without enzyme activity after pMAL-c2x and DH5 α/pMAL-c2x Fiber differentiations, engineering bacteria has all given expression to active soluble g APDH-
ChSase ABC I fusion proteins.
It can be found that using chondroitin sulfate A (CSA) and B as substrate, the engineering of expression pMAL-c2x-GAPDH-ChSase ABC I
In bacterium, E.coli JM109 expression products have highest enzyme activity, therefore best host is E.coli JM109.
Albumen to best host E.coli JM109 expression and carry out SDS-PAGE electrophoresis, take after above-mentioned ultrasonication from
30 μ l of supernatant (crude product) obtained by the heart do soluble protein component SDS-PAGE electrophoresis, and gained is centrifuged after taking above-mentioned ultrasonication
Precipitation do insoluble albumen component SDS-PAGE electrophoresis.The results are shown in Figure 4, and M is that marker (from top to bottom divides in Fig. 4
Son amount is 250kDa, 150kDa, 100kDa, 70kDa, 55kDa, 40kDa, 30kDa, 20kDa, 15kDa successively), swimming lane 1 is
Blank control, swimming lane 2 and 3 are JM109/pMAL-c2x-ChSase ABC I expression products (110kDa), swimming lane 4 and 5 is
JM109/pMAL-c2x-GAPDH-ChSase ABC I expression products (149kDa) are ChSase ABC I at arrow meaning, melt
Hop protein GAPDH-ChSase ABC I.
Statistics supernatant in GAPDH-ChSase ABC I account for total expression quantity 70% (total expression quantity be supernatant in GAPDH-
GAPDH-ChSase ABC I in ChSase ABC I and precipitation, are inactive form in precipitation), therefore, the present invention passes through
The soluble protein that fusion protein is realized that chondroitin sulfate A (CSA) BC enzymes are 70% activated above in Escherichia coli, correctly folded for the first time
Form is expressed, and subsequent purification program is greatly saved.
Embodiment 2 passes through Ni column purification chondroitin sulfate A (CSA) BC enzyme fusion proteins GAPDH-ChSase ABC I
The present embodiment realizes that affine absorption realizes that a step detaches using Ni columns.
Specific affine separating step is as follows：Thalline 50mL when by final concentration of 0.5mM IPTG induced expressions 24,
6000rpm is centrifuged 10 minutes；The phage control of non-induced expression is set simultaneously.Then it is operated respectively by following two schemes：
It is washed twice, is resuspended with column equilibration liquid Column buffer (20mM Tris-HCl, 200mM NaCl, pH7.4)
In 30mL Column buffer, ultrasonication (output power 300W, each ultrasound 5 seconds and intermittently 6 seconds, processing are carried out
Total time is 15 minutes).
The affine splitters of Ni that centrifuged supernatant is pre-equilibrated with 1mL/min by 1mL, by elution and are received
Collection.
Target protein is after Ni columns adsorb and elute.The results are shown in Figure 5, shows by one step of Ni columns target after purification
Albumen can account for 90% or more.In Fig. 5, swimming lane M be marker (from top to bottom molecular weight be successively 250kDa, 150kDa,
100kDa, 70kDa, 55kDa, 40kDa, 30kDa), swimming lane 1-2 is respectively ChSase ABC I and separated GAPDH-
ChSase ABC I。
The step that the present invention realizes the fusion protein by affine separation purifies, with the milt egg used in the prior art
White precipitation is (with reference to ANA LYDIA TKALEC, DOMINIQUE FINK et al., Isolation and Expression in
Escherichia coli of cslA and cslB,Genes Coding for the Chondroitin Sulfate-
Degrading Enzymes Chondroitinase AC and Chondroitinase B,Respectively,from
Flavobacterium heparinum.APPL.ENVIRON.MICROBIOL.2000,66(1):29-35) and a step column purification
Method compares (referring to table 1), can greatly shorten the time that purifying needs, and purifies equipment used and significantly reduce, and reduces
The production cost of purified chondroitin sulfates enzyme.It only needs to carry out or so 2 hours using the purifying of amylose resin, you can obtain
It must can be used in industrial chondroitinase ABC fusion protein.Purity reaches adaptable 95% or more.
1 protamine precipitation of table is compared with a step column purification method of the invention
After bacterial strain optimizes, e. coli jm109 (pMAL-c2x-GAPDH-ChSase ABC I) is cultivated at 37 DEG C
After 2.5 hours, 0.5mM IPTG are added, 28 DEG C of inducing temperature, the chondroitin sulfate A (CSA) BC enzyme enzyme activity of production is up to 20000IU/L
Zymotic fluid (using chondroitin sulfate A (CSA) as substrate).The chondroitin sulfate A (CSA) BC enzyme fusion proteins obtained herein become with original from common
The chondroitin sulfate A (CSA) BC enzymes of shape bacillus are compared, and have higher activity, concrete outcome as shown in table 2.
Table 2
[1]：Pay the screening of quiet rue production chondrosulphatase bacterial strain, fermentation, enzyme isolate and purify and property Quality Research [D]
Chinese Marine University, 2012.
[2]：Tao Ke, Wang Zhongyan, state's brocade beautiful jade, Hu Cheng, Deng Lin, Liu Shigui, Dai Qilin chondroitin sulfates crack ABC enzymes and generate
The screening of bacterium and Study on Fermentation [J] China antibiosis rope magazine, 2004,29 (3):138-141.
[3]:The screening of Yan Haolin, He Hanzhou, Cai Sulan, Wang Qi chondrosulphatase producing strains and isolating and purifying for enzyme
[J] microorganism journals, 2004,44 (1):79-82.
The sulphur that the present invention will play a significant role in the production of chondroitin sulfate A (CSA) BC enzymes, and produce through the invention
Aching and limp ossein ABC enzyme fusion proteins can be equally applicable to production practices with chondroitin sulfate A (CSA) BC enzymes.
Embodiment 3, using embodiment 2 prepare chondroitin sulfate A (CSA) BC enzymes production low-molecular weight chondroitin sulfate A, B or
C
It is 90% chondroitin sulfate A (CSA) BC enzymes that purity is obtained in embodiment 2, and the present embodiment uses the chondroitin sulfate of the purification
ABC enzymes produce the chondroitin sulfate A (CSA) of low molecular weight, B or C.
Specific production method is as follows：Compound concentration is the chondroitin sulfate A (CSA) of 100g/L, the solution 1L of B and C, sulfuric acid respectively
Chondroitin A, B and C molecular weight are 50kDa, derive from ox cartilaginous tissue (being purchased from Nanjing Olympic Duo Fu Buddhist nun bio tech ltd),
As reaction substrate, 10-20ml chondrosulphatase (13922IU/L) after purification is added.Reaction temperature is 30-55 DEG C, every
New enzyme solution 15mL is added in 1h, while reaction product is measured by sampling, reaction time 5-10h.
Chondroitin sulfate A (CSA), B and C are after the degradation of chondroitin sulfate A (CSA) BC enzymes, chondroitin sulfate obtained at different times
The molecular weight of A, B and C are different.As time increases, chondroitin sulfate A (CSA), B and C molecular weight is smaller and smaller, is finally reached after 10h
To 2kDa.
It describes the preferred embodiment of the present invention comprehensively above, but various alternatives and modifications can be carried out to them.Therefore,
Above description be reference should not be made to determine the scope of the present invention, but should refer to the appended claims and its whole equivalents to determine
Determine the scope of the present invention.Any feature, (being whether preferred) can be with any other feature (being whether preferred) phases
In conjunction with.Claims of the present invention is understood not to the limitation with method+function, unless leading in a certain claim
It crosses term " ... method " and clearly enumerates such limitation.
Claims (10)
1. a kind of method preparing chondroitin sulfate A (CSA) BC enzyme fusion proteins, wherein the method includes：
1) clone and connect chondroitin sulfate A (CSA) BC enzyme fusion proteins genes, clone respectively the chondroitin sulfate A (CSA) BC enzyme genes and
Glyceraldehyde 3-phosphate dehydro-genase protein gene, and connect said gene so that the glyceraldehyde 3-phosphate dehydro-genase egg subsequently expressed
It is connected in vain with chondroitin sulfate A (CSA) BC zymoprotein N-terminals；
2) recombinant expression carrier containing the chondroitin sulfate A (CSA) BC enzyme fusion proteins genes is obtained, the step 1) is connected
Chondroitin sulfate A (CSA) BC enzyme fusion proteins genetic recombination to expression vector；
3) the chondroitin sulfate A (CSA) BC enzyme fusion proteins are expressed, contain chondroitin sulfate A (CSA) BC enzymes by what is cloned in the step 2)
The expression vector of antigen-4 fusion protein gene is transformed into host cell, is expressed；And
4) harvest and purify the chondroitin sulfate A (CSA) BC enzyme fusion proteins of expression.
2. according to the method described in claim 1, wherein, chondroitin sulfate A (CSA) BC enzyme fusion proteins genes are compiled in the step 1)
Code SEQ ID NO:Amino acid sequence shown in 3.
3. according to the method described in claim 1, wherein, chondroitin sulfate A (CSA) BC enzyme fusion proteins bases are expressed in the step 2)
The expression vector of cause includes pMAL-c2x.
4. according to the method described in claim 1, wherein, the expression chondroitin sulfate A (CSA) BC enzymes merge egg in the step 3)
White host cell includes E.coli JM109 and DH5 α；
Wherein, the expression chondroitin sulfate A (CSA) BC enzyme fusion proteins conditions are that the 37 DEG C of cultures 3 of TB culture mediums are small in the step 3)
When, it is added final concentration of 0.5mM IPTG, 10-42 DEG C of Fiber differentiation 15-28 hours.
5. according to the method described in claim 1, wherein, the expression chondroitin sulfate A (CSA) BC enzymes merge egg in the step 3)
Informal voucher part is that 37 DEG C of TB culture mediums are cultivated 3 hours, final concentration of 0.5mM IPTG is added, 28 DEG C induce 24 hours.
6. the chondroitin sulfate A (CSA) BC enzyme fusion proteins prepared according to any one of Claims 1 to 5 the method.
7. a kind of DNA, the DNA sequence encoding SEQ ID NO:Amino acid sequence shown in 3.
8. a kind of recombinant vector, the recombinant vector includes the DNA sequence dna described in carrier and claim 7, wherein the carrier
Including pMAL-c2x.
9. a kind of transformant, the transformant is to import recombinant vector according to any one of claims 8 obtained from host cell to turn
Change body, wherein the host cell includes E.coli Top10, JM109, BL21, BL21 (DE3) and DH5 α.
10. a kind of method of production low-molecular weight chondroitin sulfate A, B or C, the method includes：
1) clone and connect chondroitin sulfate A (CSA) BC enzyme fusion proteins genes, clone respectively the chondroitin sulfate A (CSA) BC enzyme genes and
Glyceraldehyde 3-phosphate dehydro-genase protein gene, and connect said gene so that the glyceraldehyde 3-phosphate dehydro-genase egg subsequently expressed
It is connected in vain with chondroitin sulfate A (CSA) BC zymoprotein N-terminals；
2) recombinant expression carrier containing the chondroitin sulfate A (CSA) BC enzyme fusion proteins genes is obtained, the step 1) is connected
Chondroitin sulfate A (CSA) BC enzyme fusion proteins genetic recombination to expression vector；
3) the chondroitin sulfate A (CSA) BC enzyme fusion proteins are expressed, contain chondroitin sulfate A (CSA) BC enzymes by what is cloned in the step 2)
The expression vector of antigen-4 fusion protein gene is transformed into host cell, is expressed；
4) harvest and purify the chondroitin sulfate A (CSA) BC enzyme fusion proteins of expression；And
5) the chondroitin sulfate A (CSA) BC enzyme fusion proteins degradation substrate of the step 4) purifying is used, reaction temperature is 30-55 DEG C,
Reaction time is 5-10h, production low-molecular weight chondroitin sulfate A, B or C.
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