CN109897812B - Recombinant bacterium for expressing chondroitin 4-sulfate transferase gene and application thereof - Google Patents
Recombinant bacterium for expressing chondroitin 4-sulfate transferase gene and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant bacterium for expressing chondroitin 4-sulfate transferase gene and application thereof, belonging to the technical field of biological engineering. According to the invention, Escherichia coli or Bacillus subtilis is taken as a host, chondroitin 4-sulfate transferase gene with an amino acid sequence shown as SEQ ID NO.1 is expressed, four recombinant bacteria are constructed, the high-efficiency expression of chondroitin 4-sulfate transferase is realized, and finally the enzyme activities respectively reach 41.3U/mL, 32.8U/mL, 36.5U/mL and 50.2U/mL. The enzyme is used as a catalyst, chondroitin is used as a substrate, and the chondroitin sulfate A is synthesized through one-step catalysis, so that the final conversion efficiency reaches 80%. The method of the invention has potential and very wide value in the industrial application of the synthesis of the chondroitin sulfate A.
Description
Technical Field
The invention relates to a recombinant bacterium for expressing chondroitin 4-sulfate transferase gene and application thereof, belonging to the technical field of biological engineering.
Background
Chondroitin Sulfate (CS) is a straight-chain acidic mucopolysaccharide formed by alternately connecting glucuronic acid (D-GlcUA) and N-acetylgalactosamine (GalNAc) by beta-1, 3 bonds and beta-1, 4 bonds, and the sulfated structure of the Chondroitin sulfate has various forms, and the main medicinal components at present are A and C isomers. Chondroitin Sulfate A (CSA) has various biological activities and medicinal values, is clinically used for treating rheumatism and arthritis, can endow cartilage gel-like characteristics and anti-deformation capability to a human body, is called soft gold, can be used as a dietary supplement and a humectant, and is widely applied to the fields of food and cosmetics.
At present, the industrialized production method of chondroitin sulfate A is an animal tissue extraction method, namely, the chondroitin sulfate A is prepared from animal cartilages such as pigs, cattle, sharks and the like by adopting process methods such as alkaline hydrolysis, protease enzymolysis and the like. However, the extraction method has many problems, such as limited raw materials, complex production process, unstable product quality, low industrial level, serious environmental pollution and the like, and the development of the chondroitin sulfate A industry is severely restricted.
Therefore, researchers at home and abroad continuously search for a method for efficiently producing chondroitin sulfate A by using a biological method. The method for synthesizing chondroitin sulfate A by the enzyme method has the characteristics of high selectivity, high efficiency, no toxicity, low pollution and the like, and gradually receives wide attention of people. Chondroitin sulfate A can be synthesized in one step by catalysis of chondroitin 4-sulfate transferase (C4ST), and at present, chondroitin 4-sulfate transferase is mainly extracted from animal cells, so that the cost is high and the industrial production is not facilitated. HeWenqin et al have succeeded in heterologous expression of H.sapiens-derived C4ST in P.pastoris host by plasmid ligation of pJK912 (HeWenqin, Applied Microbiology Biotechnology,2017,101: 6919-. Therefore, how to obtain high yield of C4ST becomes the first problem for efficient synthesis of chondroitin sulfate A.
Disclosure of Invention
The recombinant Escherichia coli for expressing the chondroitin 4-sulfate transferase gene is finally obtained by screening the expression system, the enzyme activity of C4ST secreted and expressed by the recombinant Escherichia coli is as high as 50.2U/mL, and conditions are created for efficient synthesis of chondroitin sulfate A.
The first purpose of the invention is to provide a recombinant bacterium for expressing chondroitin 4-sulfate transferase, which takes escherichia coli or bacillus subtilis as a host to express chondroitin 4-sulfate transferase (C4ST) gene with amino acid sequence shown as SEQ ID NO. 1.
In one embodiment of the present invention, the nucleotide sequence of chondroitin 4-phosphotransferase (C4ST) is represented by SEQ ID NO. 4.
In one embodiment of the invention, the recombinant bacterium is expressed in bacillus subtilis by taking pHT01 as an expression vector.
In one embodiment of the invention, the recombinant bacteria are expressed in escherichia coli by respectively using pET22a, pET20b or pTrcHisA as expression vectors.
In one embodiment of the invention, the recombinant bacterium is obtained by connecting the C4ST gene with the maltose fusion protein gene through an oligopeptide linker GGGS gene, and then connecting the gene with an expression vector.
In one embodiment of the invention, the nucleotide sequence of the oligopeptide linker GGGS gene is shown as SEQ ID No. 2.
In one embodiment of the present invention, the nucleotide sequence of the maltoglycoprotein MBP gene is shown in SEQ ID NO. 3.
The second purpose of the invention is to provide a construction method of the recombinant bacterium, which is to connect maltose fusion protein, oligopeptide linker GGGS, chondroitin 4-sulfate transferase C4ST gene in sequence, then connect to an expression vector to obtain a recombinant plasmid, and then transform the recombinant plasmid into a host bacterium.
In one embodiment of the present invention, the construction method comprises the following steps:
(1) c4ST gene with amino acid sequence shown as SEQ ID NO.1 is connected with maltose fusion protein MBP gene through oligopeptide linker GGGS gene with nucleotide sequence shown as SEQ ID NO.2 to obtain fusion protein MBP-C4ST gene;
(2) the MBP-C4ST gene is respectively connected with plasmids pHT01, pET22a, pET20b and pTrcHisA to obtain recombinant plasmids pHT01-MBP-C4ST, pET22a-MBP-C4ST, pET20b-MBP-C4ST and pTrcHisA-MBP-C4 ST;
(3) and (3) respectively transforming the obtained recombinant plasmids into host bacteria B.subtilis or E.coli to obtain recombinant bacteria: subtilis168-pHT01-MBP-C4ST and E.coli BL21-pET22a-MBP-C4ST, E.coli BL21-pET20b-M BP-C4ST, E.coli BL21-pTrcHisA-MBP-C4 ST.
The third object of the present invention is to provide a method for producing chondroitin 4-sulfatase C4ST, wherein the recombinant bacterium is used for fermentation production.
The fourth purpose of the invention is to provide a method for producing chondroitin sulfate A, which is characterized in that the method is used for fermentation production by using crude enzyme liquid of the recombinant bacteria and using chondroitin as a substrate.
In one embodiment of the invention, the fermentative production is carried out in a sulfate donor adenosine 3 '-phosphate-5' -phosphosulfate (PAPS) regeneration system.
In one embodiment of the present invention, the PAPS regeneration system includes: 3 '-phosphoadenosine-5' -phosphate (PAP), 3 '-phosphoadenosine-5' -phosphate sulfate (PAPS), and acyl phosphotransferase (AST IV).
In one embodiment of the present invention, the system of the PAPS regeneration system is: 20-100mM Tris-HCl (pH 7.0), 1-100mM PAP, 1-300mM PNPS, 0.1-10mL AST IV crude enzyme solution.
In one embodiment of the present invention, the method for producing chondroitin sulfate a specifically comprises: adding 1-100mM PAP, 1-300mM PNPS, 0.1-10mL AST IV crude enzyme solution, 0.1-5mL C4ST crude enzyme solution into 20-100mM Tris-HCl solution with pH 7.0, and reacting at 20-70 deg.C for 20-60 h.
The invention also provides application of the recombinant bacterium in the field of pharmacy.
The invention has the beneficial effects
The invention adopts different plasmids and vectors to express chondroitin 4-sulfate transferase, and four recombinant bacteria B.subtiliss 168-pHT01-MBP-C4ST and E.coli BL21-pET22a-MBP-C4ST, E.coli BL21-pET20b-MBP-C4ST and E.coli BL21-pTrcHisA-MBP-C4ST are constructed, so that the high-efficiency expression of the chondroitin 4-sulfate transferase is realized, the final enzyme activities respectively reach 41.3U/mL, 32.8U/mL, 36.5U/mL and 50.2U/mL, the enzyme is used as a catalyst, the chondroitin is used as a substrate to synthesize the sulfate A in a one-step catalytic manner, and the final conversion efficiency reaches 80%. The method of the invention has potential and very wide value in the industrial application of the synthesis of the chondroitin sulfate A.
Drawings
FIG. 1: enzyme activity comparison of recombinant expression chondroitin-4-sulfate transferase. 1: strain b. subtilis168-pHT01-MBP-C4ST, 2: coli BL21-pET22a-MBP-C4ST, 3: coli BL21-pET20b-MBP-C4ST, 4: coli BL21-pTrcHisA-MBP-C4 ST;
FIG. 2: and (3) performing high performance liquid chromatogram of the reaction liquid of the CSA after carrying out enzymolysis on chondroitinase ABC.
Detailed Description
Related nucleotide sequence information in the sequence listing:
(1) the sequence of SEQ ID NO.1 is an amino acid sequence of chondroitin 4-sulfate transferase derived from animals;
(2) the sequence of SEQ ID NO.2 is the nucleotide sequence of oligopeptide linker GGGS gene;
(3) the SEQ ID NO.3 sequence is a nucleotide sequence of maltose fusion protein derived from Escherichia coli;
(4) the sequence information of SEQ ID NO.4 is a nucleotide sequence of chondroitin and 4-sulfotransferase derived from animals;
(5) the sequence information of SEQ ID NO.5 is a nucleotide sequence of the sulfur transferase derived from animal cells;
example 1: construction of recombinant expression vectors
(1) Obtaining of fusion protein MBP-C4ST gene fragment
In order to allow the C4ST gene derived from animal cells to be expressed efficiently in microbial cells, Maltose Binding Protein (MBP) derived from escherichia coli needs to be fused to the N-terminus of the enzyme.
The MBP gene with a nucleotide sequence shown as SEQ ID NO.3 is artificially synthesized and is used as a template, primers MBP-S and RH-MBP-C4ST-A are used for amplification to obtain the MBP gene, meanwhile, a C4ST gene with a nucleotide sequence shown as SEQ ID NO.4 is artificially synthesized and is used as a template, primers RH-MBP-C4ST-S and C4ST-A are used for amplification to obtain a C4ST gene, then the MBP gene and the C4ST gene are fused by adopting a fusion PCR technology, and finally, the primers MBP-S and C4ST-A are used for amplification to obtain a fusion protein MBP-C4ST gene.
(2) Construction of pHT01-MBP-C4ST
And (2) taking the MBP-C4ST gene obtained in the step (1) as a template, amplifying by using primers pHT-01-S and pHT-01-A to obtain an MBP-C4ST gene, and then integrating the MBP-C4ST gene on a pHT01 plasmid by using a one-step homologous recombination kit to obtain a recombinant vector pHT01-MBP-C4 ST.
(3) Construction of pET22a-MBP-C4ST
And (2) taking the MBP-C4ST gene obtained in the step (1) as a template, amplifying by using primers pET22a-S and pET22a-A to obtain an MBP-C4ST gene, and integrating the MBP-C4ST gene onto a pET22a plasmid by using a one-step homologous recombination kit to obtain a recombinant vector pET22a-MBP-C4 ST.
(4) Construction of pET20b-MBP-C4ST
And (2) taking the MBP-C4ST gene obtained in the step (1) as a template, amplifying by using primers pET20b-S and pET20b-A to obtain an MBP-C4ST gene, and integrating the MBP-C4ST gene onto pET20b plasmid by using a one-step homologous recombination kit to obtain a recombinant vector pET20b-MBP-C4 ST.
(5) Construction of pTrcHisA-MBP-C4ST
And (2) taking the MBP-C4ST gene obtained in the step (1) as a template, amplifying by using primers pTrcHisA-S and pTrcHisA-A to obtain an MBP-C4ST gene, and integrating the MBP-C4ST gene onto a pTrcHisA plasmid by using a one-step homologous recombination kit to obtain a recombinant vector pTrcHisA-MBP-C4 ST.
TABLE 1 primer sequence Listing
Example 2: construction of engineered Strain
(1) Construction of recombinant Bacillus subtilis
The B.subtilis168 single colony is inoculated into a test tube containing 5mL of GM I solution, and is placed at 30 ℃ for shaking culture at 200rpm for 16h to obtain a first seed solution. 2mL of the first seed solution was transferred to 18mL of fresh GM I solution and cultured at 37 ℃ for 3.5h at 200rpm to obtain a second seed solution. Transferring 10mL of the second seed solution into 90mL of GM II solution, culturing at 37 ℃ and 200rpm for 90min, centrifuging at 4 ℃ for 10min at 5,000g, collecting thallus, removing supernatant, and reserving 10mL of liquid for suspending thallus, wherein the suspended thallus is competent cells which can be directly used for heat shock transformation (45 ℃, 90S). Proper amount of recombinant plasmid pHT01-MBP-C4ST is added into B.subtilis168 competent cells, after the cells are cultured at 37 ℃ and 200rpm for 90min, an LB culture medium with Chl resistance is coated, and the engineering strain which is verified to be correct is named as B.subtilis168-pHT01-MBP-C4 ST.
(2) Construction of recombinant Escherichia coli
Coli BL21 single colonies were picked and inoculated into a flask containing 25mL LB liquid medium, cultured at 37 ℃ and 200rpm for 8h, and then transferred with an inoculum size of 2%. When the concentration of the cells reaches about 0.48, competent cells are prepared. The cells were centrifuged at 6000rpm for 5min at 4 ℃ to collect the cells, and the supernatant was removed. After the cells were washed three times with 10% glycerol, the cells were transformed by electric shock, and appropriate amounts of recombinant plasmids pET22a-MBP-C4ST, pET20b-MBP-C4ST and pTrcHisA-MBP-C4ST were added, respectively. After culturing at 37 ℃ and 200rpm for 60min, coating corresponding resistance (Kan, Amp) screening culture media respectively, and verifying that correct engineering strains are named as E.coli BL21-pET22a-MBP-C4ST, E.coli BL21-pET20b-MBP-C4ST and E.coli BL21-pTrcHisA-MBP-C4 ST.
Example 3: construction of 3 '-adenosine phosphate-5' -phosphosulfate PAPS regeneration system
Since the C4ST reaction system requires a sulfate group donor, namely, 3 '-phosphoadenosine-5' -phosphosulfate PAPS, the acyl phosphotransferase AST IV can be expressed and obtained according to the method described in the literature (Yuh-selling Yang, Protein Expression and Purification,1996,8: 423-429), and a regeneration system of 3 '-phosphoadenosine-5' -phosphosulfate PAPS can be constructed. The specific operating steps are given below:
(1) expression of acyl-phosphotransferase AST IV
The acyl sulfate transferase gene AST IV used in the invention is derived from an animal mouse. The plasmid pTrcHisA is used as an expression vector, the artificially synthesized gene fragment AST IV with the nucleotide sequence shown as SEQ ID NO.5 is used as a target fragment, and the expression vector pTrcHisA-AST IV is constructed by an enzyme digestion connection method. The expression vector pTrcHisA-AST IV is transferred into E.coli BL21 to obtain a recombinant strain E.coli BL21-pTrcHisA-AST IV.
Selecting a single colony of a recombinant strain E.coli BL21-pTrcHisA-ASTIV, inoculating the single colony into an LB liquid culture medium, culturing at 37 ℃ and 200r/min for 10 hours, and then transferring, wherein the transferring amount is 5 percent when OD is reached600When the concentration is about 0.8, 0.4mM IPTG is used for induction, and the induction time is 8 h. And collecting thalli after the shake flask culture is finished, carrying out ultrasonic crushing, and centrifuging to obtain a supernatant, namely the AST IV crude enzyme solution.
(2) Construction of 3 '-adenosine phosphate-5' -phosphosulfate (PAPS) regeneration System
The PAPS regeneration system includes: 50mM Tris-HCl (pH 7.0), 1mM PAP, 5mM 3 '-adenosine phosphate-5' -phosphosulfate (PNPS), 0.3mL AST IV crude enzyme solution, and reacted at 37 ℃ for 24 hours.
TABLE 2 primer sequence Listing
Example 4: expression and enzyme activity determination of C4ST
(1) Expression of C4ST in recombinant Bacillus subtilis
Basal fermentation medium (g/L): glucose 10, yeast extract 15, Na2HPO4·12H2O 1，MgSO 4 1，NaCl 8，pH 7.0。
Lysis buffer (mM): NaCl100, NaH2PO450，Na2HPO450，pH 7.5。
Selecting an engineering strain B.subtilis168-pHT01-MBP-C4ST, inoculating the engineering strain B.subtilis168-pHT01-MBP-C4ST into LB culture medium added with chloramphenicol for overnight culture, culturing at 37 ℃ and 200rpm for 12-14 h, inoculating the engineering strain into a basic fermentation culture medium according to the inoculation amount of 3% when the OD value reaches 0.6-0.8, continuously culturing at 37 ℃ and 200rpm for 16-20 h, centrifuging the obtained bacterial liquid at low temperature (4 ℃, 8000rpm, 4-5 min), and discarding supernatant to collect precipitated bacteria. Adding lysozyme into the lysis buffer solution to a final concentration of 2mg/mL, and washing and resuspending the thallus collected in the previous step. The cell suspension was sonicated for 15min (power 200W, sonication 1s, pause 2 s). And (3) centrifuging the cell disruption solution at low temperature (4 ℃, 10000rpm, 20min), and obtaining supernatant, namely the C4ST crude enzyme solution.
(2) Expression of C4ST in recombinant E.coli
The engineering strains E.coli BL21-pET22a-MBP-C4ST, E.coli BL21-pET20b-MBP-C4ST and E.coli BL21-pTrcHisA-MBP-C4ST are picked, and single colonies are respectively inoculated into LB liquid culture medium added with antibiotics for overnight culture. Then transferring the strain into LB culture medium containing 100mL, wherein the transferring amount is 2%, and when the concentration of the strain reaches 0.48, inducing the strain by IPTG at the induction temperature of 25 ℃ for 8 h. After induction, centrifuging for 5min at 4 ℃ and 8000rpm, washing the thallus for several times by using PBS buffer solution, ultrasonically crushing the thallus for 15min (power 400W, ultrasonic for 2s and intermittent for 3s), and collecting supernatant after centrifugation, namely C4ST crude enzyme solution.
(3) Enzyme activity assay of C4ST
The enzyme activity determination method of the chondroitin 4-sulfate transferase comprises the following steps: 1-10mg/mL chondroitin, 1-300mM PNPS, 1-100mM PAP, 0.1-10mL AST IV crude enzyme solution, 0.1-5mL C4ST crude enzyme solution, and 50mM Tris-HCl solution with pH 7.0. The reaction conditions are as follows: reacting at 20-70 deg.C for 20-60 hr, boiling for 10min, and finishing reaction. Centrifuging the reaction liquid, collecting supernatant, and measuring the light absorption value of the p-nitrophenol at the wavelength of 400 nm. 1U C4ST defines the amount of enzyme required to produce 1. mu.M/mL of p-nitrophenol per hour.
Chondroitin sulfate A is synthesized in one step by taking chondroitin as a substrate and carrying out a catalytic experiment. The reaction system is as follows: 10mg/mL chondroitin, 15mM PNPS, 5mM PAP, 0.3mL AST IV crude enzyme, 0.2mL C4ST crude enzyme, 50mM Tris-HCl solution with pH 7.0. The reaction conditions are as follows: reacting at 37 ℃ for 24h, and boiling for 10min to finish the reaction. Centrifuging the reaction liquid, collecting supernatant, and measuring the light absorption value of the p-nitrophenol at the wavelength of 400 nm. According to the enzyme activity determination of C4ST, the result is shown in figure 1, when E.coli BL21 is taken as an expression host and pTrcHisA is taken as an expression vector, the enzyme activity of C4ST is the highest and reaches 50.2U/mL.
TABLE 3 enzymatic Activity of C4ST expressed by different expression systems
Example 5: enzyme activity determination of C4ST from different sources
Referring to the construction method of E.coli BL21-pTrcHisA-MBP-C4ST in example 2, the source of C4ST sequence was changed from Mus musculus (mouse) (SEQ ID NO:1) to Sus scrofa (pig) (NCBI accession No. 100519444) and Callorhinchsimii (shark) (NCBI accession No. 103175681), recombinant strains E.coli BL21-pTrcHisA-MBP-C4ST (pig) and E.coli BL21-pTrcHisA-MBP-C4ST (shark) were constructed, and the enzyme activities of C4ST were expressed and measured, respectively, according to the method of example 4, and the results were 50.2, 27.3 and 39.5U/mL, respectively, as shown in Table 4.
TABLE 4 enzymatic Activity for heterologous expression of C4ST from different sources
Example 6: production of chondroitin sulfate A by recombinant bacterium E.coli BL21-pET22a-MBP-C4ST
The preparation method of the CSA comprises the following steps: and (3) constructing an enzyme catalytic reaction system by using chondroitin as a substrate to synthesize the CSA. The reaction system comprises: to a 50mM Tris-HCl solution having a pH of 7.0, 5mM PAP, 15mM PNPS, 0.3mL AST IV crude enzyme solution, 0.2mL C4ST crude enzyme solution (crude enzyme solution produced in E.coli BL21-pET22a-MBP-C4ST obtained in step (1) of example 4), 10mg/mL chondroitin was added, mixed, reacted in a 37 ℃ water bath for 24 hours, and boiled for 10 minutes to complete the reaction.
The CSA detection method comprises the following steps: centrifuging the reaction solution prepared from the CSA, adding chondroitin sulfate ABC enzyme solution (which can decompose the CSA into 4-sulfated disaccharide, 4S), mixing, reacting in 37 deg.C water bath for 1 hr, boiling for 10min, and finishing reaction. Centrifuging to obtain supernatant, filtering with 0.45 μm filter membrane, and detecting disaccharide component by high performance liquid chromatography using the filtrate as sample.
Measuring the CSA enzymolysis reaction liquid by using a high performance liquid chromatography, wherein the chromatographic conditions are as follows: chromatographic column Hypersil SAX column (250 mm. times.4.6 mm, 5 μ M), detection wavelength of 232nm, mobile phase A of deionized water, and mobile phase B of 2M/L sodium chloride solution at flow rate of 1 mL/min.
The catalytic efficiency of C4ST was: y ═ 18.68 (A)1-A0)+0.15]*n*10-3。A0To control the absorbance, A1The absorbance value of the enzyme activity of C4ST is measured, and n is the dilution factor.
The detection of the CSA preparation solution revealed (as shown in fig. 2) that 4-sulfated disaccharide (4S), which is one of disaccharide units of chondroitin sulfate a, can exist stably, which indicates that the enzymatic synthesis of chondroitin sulfate a has succeeded by using chondroitin as a substrate, and the catalytic efficiency has reached 80% or more.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
WUXI CHENMING BIOTECHNOLOGY Co.,Ltd.
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acgcagaagt acaacacctc cttccacaag cgctacggca ccaagatcat ccgacgccag 660
cggaagaacg ccacgcagga ggccctgcgc aagggggacg atgtcaagtt cgaggagttc 720
gtggcctacc tcatcgaccc ccacacccag cgggaggagc ccttcaacga gcactggcag 780
acggtctact ctctctgcca cccgtgccac atccactacg acctcgtggg caagtatgag 840
acactggagg aggactccaa ttacgtactg cagctggccg gagtgagcgg ctacctgaag 900
ttccccacct atgcaaagtc cacccgaact accgacgaga tgaccacgga gttcttccag 960
aacatcagcg ccgagcacca gacacagctg tacgaagtct acaaactgga ctttttaatg 1020
ttcaactact cagtgccaaa ctacctgaag ttggattag 1059
<210> 5
<211> 876
<212> DNA
<213> Artificial Synthesis
<400> 5
atggaattca gccgtccacc gctggtgcat gtgaagggca tcccgctgat caagtacttt 60
gcggagacga tcggcccgct gcagaatttt accgcgtggc cggatgatct gctgatcagc 120
acctacccga agagcggtac gacgtggatg agtgagattc tggacatgat ctatcaaggc 180
ggcaagctcg agaaatgcgg tcgcgcccca atctatgccc gcgttccgtt tctggaattc 240
aagtgcccgg gcgtgccgag cggtctggaa acgctggaag aaaccccagc gccacgtctg 300
ctgaaaacgc atctgccact cagtctgctg ccgcagagtc tgctggatca gaaggtgaaa 360
gtgatctaca tcgcccgcaa cgccaaagac gtggtggtta gctactacaa tttttacaac 420
atggccaagc tccacccaga tccgggcacg tgggacagct ttctggagaa tttcatggac 480
ggcgaggtta gctacggtag ctggtaccag cacgtgaaag aatggtggga actgcgccat 540
acgcatccgg tgctgtatct gttctacgag gacatcaagg agaacccgaa gcgcgagatc 600
aaaaagattc tggagttcct cggtcgcagt ctgccggagg agacggttga tagcatcgtg 660
caccacacga gcttcaagaa gatgaaagaa aattgcatga ccaattacac cacgatcccg 720
accgaaatca tggaccacaa cgtgagcccg ttcatgcgta aaggcacgac cggcgactgg 780
aagaacacct ttaccgttgc ccagaacgaa cgcttcgatg cgcactacgc caagaccatg 840
accgactgcg acttcaagtt ccgctgcgag ctgtaa 876
<210> 6
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 6
atgaaaataa aaacaggtgc acg 23
<210> 7
<211> 47
<212> DNA
<213> Artificial Synthesis
<400> 7
cgcagactcg tatcaccaag ggtggtggtt ctatgaagcc ggcgctg 47
<210> 8
<211> 47
<212> DNA
<213> Artificial Synthesis
<400> 8
cagcgccggc ttcatagaac caccaccctt ggtgatacga gtctgcg 47
<210> 9
<211> 25
<212> DNA
<213> Artificial Synthesis
<400> 9
ctaatccaac ttcaggtagt ttggc 25
<210> 10
<211> 45
<212> DNA
<213> Artificial Synthesis
<400> 10
caccggaatt agcttggtac catgaaaata aaaacaggtg cacgc 45
<210> 11
<211> 46
<212> DNA
<213> Artificial Synthesis
<400> 11
tatgttacaa tagctggtac cctaatccaa cttcaggtag tttggc 46
<210> 12
<211> 45
<212> DNA
<213> Artificial Synthesis
<400> 12
gtggtggtgg tggtgctcga gatgaaaata aaaacaggtg cacgc 45
<210> 13
<211> 46
<212> DNA
<213> Artificial Synthesis
<400> 13
aagcttgcgg ccgcactcga gctaatccaa cttcaggtag tttggc 46
<210> 14
<211> 45
<212> DNA
<213> Artificial Synthesis
<400> 14
gtggtggtgg tggtgctcga gatgaaaata aaaacaggtg cacgc 45
<210> 15
<211> 46
<212> DNA
<213> Artificial Synthesis
<400> 15
aagcttgcgg ccgcactcga gctaatccaa cttcaggtag tttggc 46
<210> 16
<211> 45
<212> DNA
<213> Artificial Synthesis
<400> 16
cgatggggat ccgagctcga gatgaaaata aaaacaggtg cacgc 45
<210> 17
<211> 46
<212> DNA
<213> Artificial Synthesis
<400> 17
gtaccagctg cagatctcga gctaatccaa cttcaggtag tttggc 46
<210> 18
<211> 26
<212> DNA
<213> Artificial Synthesis
<400> 18
cgcggatcca tggagttctc ccgtcc 26
<210> 19
<211> 31
<212> DNA
<213> Artificial Synthesis
<400> 19
ccgctcgagt catagttcac aacgaaactt g 31
Claims (6)
1. A recombinant bacterium for expressing chondroitin 4-sulfate transferase is characterized in that Escherichia coli or Bacillus subtilis is used as a host to express a nucleotide sequence obtained by connecting a chondroitin 4-sulfate transferase gene with a nucleotide sequence shown as SEQ ID No.4 and a maltose fusion protein gene with a nucleotide sequence shown as SEQ ID No.3 through an oligopeptide linker GGGS gene with a sequence shown as SEQ ID No. 2.
2. The recombinant bacterium according to claim 1, wherein pHT01 is used as an expression vector.
3. The recombinant bacterium according to claim 1, wherein pET22a, pET20b or pTrcHisA is used as an expression vector.
4. A construction method of the recombinant bacterium according to claim 1, characterized in that maltose fusion protein gene, oligopeptide linker GGGS gene and chondroitin-4-sulfate transferase gene are connected in sequence, and then connected to an expression vector to obtain a recombinant plasmid, and then transformed into host bacteria; the nucleotide sequence of the maltose fusion protein gene is shown as SEQ ID NO. 3; the nucleotide sequence of the oligopeptide linker GGGS gene is shown in SEQ ID NO. 2; the nucleotide sequence of the chondroitin 4-sulfate transferase gene is shown as SEQ ID NO. 4.
5. A method for producing chondroitin 4-sulfate transferase, which comprises subjecting the recombinant bacterium according to any one of claims 1 to 3 to fermentation.
6. The application of the recombinant bacterium of claims 1-3 in the pharmaceutical field.
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| CN111621533B (en) * | 2020-06-28 | 2021-09-28 | 江南大学 | Method for efficiently producing chondroitin sulfate A by artificial enzyme method |
| CN114891760B (en) * | 2022-05-25 | 2023-06-23 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis CHST12 protein and application thereof in antibiosis |
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| CN101965196A (en) * | 2007-12-27 | 2011-02-02 | 斯特里克再生医学学院,斯特里克学院事务所 | Sugar chain-related gene and use thereof |
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| CN106755205A (en) * | 2016-11-14 | 2017-05-31 | 江南大学 | A kind of method that enzyme process prepares chondroitin sulfate |
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| CN101965196A (en) * | 2007-12-27 | 2011-02-02 | 斯特里克再生医学学院,斯特里克学院事务所 | Sugar chain-related gene and use thereof |
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