CN114958885A - Gene construction and application method capable of rapidly detecting beta-1, 3-glucan - Google Patents

Gene construction and application method capable of rapidly detecting beta-1, 3-glucan Download PDF

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CN114958885A
CN114958885A CN202210743979.6A CN202210743979A CN114958885A CN 114958885 A CN114958885 A CN 114958885A CN 202210743979 A CN202210743979 A CN 202210743979A CN 114958885 A CN114958885 A CN 114958885A
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cbm2
fusion protein
13glucan1a
13glucan1b
gene
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王瑞明
张淑玥
张子洋
汪俊卿
李丕武
苏静
王婷
梁晓丽
魏晓凤
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Qilu University of Technology
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Abstract

The invention provides a gene construction and application method capable of rapidly detecting beta-1, 3-GLUCAN, and provides a nucleotide sequence of a fusion protein 13GLUCAN1a-CBM2 coding gene shown in SEQ ID No.1, a nucleotide sequence of a fusion protein CBM2-13GLUCAN1b coding gene shown in SEQ ID No.2, and an application of the fusion protein 13GLUCAN1a-CBM2 and the fusion protein CBM2-13GLUCAN1b in detecting beta-1, 3-GLUCAN, wherein the fusion protein 13GLUCAN1a-CBM2 and the fusion protein CBM2-13GLUCAN1b provided by the invention can be specifically combined with the beta-1, 3-GLUCAN and generate fluorescence, and can be used for detecting the beta-1, 3-GLUCAN.

Description

Gene construction and application method capable of rapidly detecting beta-1, 3-glucan
Technical Field
The invention relates to the field of bioengineering, in particular to a gene construction and application method capable of rapidly detecting beta-1, 3-glucan.
Background
The beta-1, 3-glucan is an important bioactive polysaccharide, is widely distributed in plants, fungi and bacteria, and has the advantages of wide source, reproducibility, safety and the like. The unique structural characteristics of the beta-1, 3-glucan enable the beta-glucan to have multiple special physiological functions of reducing cholesterol, regulating blood sugar, improving immunity and the like, is an ideal health food, can be used as a food additive in the field of food industry, has good application value and development prospect, and is a hot spot of domestic and foreign research at present.
Chinese patent document CN105842208A (application No. 201610160837.1) discloses a method for detecting beta-glucan based on a fluorescence enhancement principle, which takes a processed silicon wafer as a substrate, deposits titanium dioxide and graphene oxide hydrogel on the silicon wafer by combining a spin coating technology to prepare titanium dioxide/graphene oxide hydrogel one-dimensional photonic crystal, and carries out gold plating on the photonic crystal by a vacuum evaporation plating method to prepare gold-plated titanium dioxide/graphene oxide hydrogel one-dimensional photonic crystal, and takes the photonic crystal as a fluorescence enhancement substrate to detect the beta-glucan by detecting the fluorescence intensity of a fluorescence composite product of the beta-glucan and aniline blue in specific combination; this method is significantly different from the detection methods provided herein.
In the prior art, few reports are provided about methods for detecting substrates by using the fusion of CBM structural domains and fluorescent proteins as probes.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a gene construction and application method capable of rapidly detecting beta-1, 3-glucan.
The inventor adds a section a of 13GLUCAN1 gene to the N end of the gene CBM2 through a Linker (connecting peptide) to construct fusion protein 13GLUCAN1a-CBM2, and adds a section b of 13GLUCAN1 gene to the C end of the CBM2 through the Linker (connecting peptide) to construct fusion protein CBM2-13GLUCAN1 b. The invention discovers that two fusion proteins 13GLUCAN1a-CBM2 and CBM2-13GLUCAN1b can generate strong fluorescence when being combined with substrates beta-1, 3-GLUCAN at the same time, and fusion proteins 13GLUCAN1a-CBM2 and CBM2-13GLUCAN1b do not generate obvious fluorescence when not being combined with the substrates, so that the fusion proteins 13GLUCAN1a-CBM2 and CBM2-13GLUCAN1b can be used for detecting the beta-1.3-GLUCAN.
The technical scheme of the invention is as follows:
a fusion protein 13GLUCAN1a-CBM2 coding gene has a nucleotide sequence shown in SEQ ID NO. 1.
A fusion protein CBM2-13GLUCAN1b encoding gene has a nucleotide sequence shown in SEQ ID NO. 2.
A recombinant vector comprises the nucleotide sequence of the 13GLUCAN1a-CBM2 coding gene, which is shown in SEQ ID NO. 1.
A recombinant vector comprises the nucleotide sequence of the CBM2-13GLUCAN1b coding gene, which is shown as SEQ ID NO. 2.
A recombinant bacterium comprises the nucleotide sequence of the 13GLUCAN1a-CBM2 coding gene, which is shown in SEQ ID NO. 1.
A recombinant strain comprises the nucleotide sequence of the CBM2-13GLUCAN1b coding gene, which is shown as SEQ ID NO. 2.
A construction method of Escherichia coli engineering bacteria containing fusion protein 13GLUCAN1a-CBM2 gene comprises the following steps:
(1) synthesizing a 13GLUCAN1a-CBM2 gene segment, wherein the nucleotide sequence of the gene segment is shown as SEQ ID NO. 1;
(2) inserting the 13GLUCAN1a-CBM2 gene fragment prepared in the step (1) into a pET-20b (+) vector to obtain a recombinant plasmid pET-20b (+) -13GLUCAN1a-CBM 2;
(3) preparing competent cells of escherichia coli BL21(DE3), carrying out enzyme digestion on the recombinant plasmid pET-20b (+) -13GLUCAN1a-CBM2 prepared in the step (2), transforming the competent cells of escherichia coli BL21(DE3), and screening positive clones to obtain the engineering bacteria of escherichia coli containing the fusion protein 13GLUCAN1a-CBM 2.
A construction method of Escherichia coli engineering bacteria containing fusion protein CBM2-13GLUCAN1b gene comprises the following steps:
synthesizing a CBM2-13GLUCAN1b gene segment, wherein the nucleotide sequence of the CBM2-13GLUCAN1b gene segment is shown as SEQ ID NO. 2;
secondly, inserting the CBM2-13GLUCAN1b gene fragment prepared in the step I into a pET-20b (+) vector to obtain a recombinant plasmid pET-20b (+) -CBM2-13GLUCAN1 b;
preparing competent cells of escherichia coli BL21(DE3), carrying out enzyme digestion on the recombinant plasmid pET-20b (+) -CBM2-13GLUCAN1b prepared in the step II, transforming the competent cells of escherichia coli BL21(DE3), and screening positive clones to obtain the engineering bacteria of escherichia coli containing the fusion protein CBM2-13GLUCAN1 b.
According to the present invention, preferably, the method for screening positive clones in step (3) or step (c) is that the transformed cells are spread on LB solid medium containing 100. mu.g/mL ampicillin, cultured at 37 ℃, a single colony is selected and inoculated into LB liquid medium containing 100. mu.g/mL ampicillin, cultured overnight at 37 ℃, then verified by PCR to obtain positive clones of the target gene band, and after sequencing, the strain with the correct sequencing result is retained as the target strain.
The Escherichia coli engineering bacteria containing the fusion protein 13GLUCAN1a-CBM2 gene constructed by the method is applied to the production of the fusion protein 13GLUCAN1a-CBM 2.
The Escherichia coli engineering bacteria containing the fusion protein CBM2-13GLUCAN1b gene constructed by the method is applied to the production of the fusion protein CBM2-13GLUCAN1 b.
Application of fusion protein 13GLUCAN1a-CBM2 coded by the nucleotide sequence shown in SEQ ID NO.1 and fusion protein CBM2-13GLUCAN1b coded by the nucleotide sequence shown in SEQ ID NO.2 in detection of beta-1, 3-GLUCAN.
A method of detecting β -1, 3-glucan comprising the steps of:
mixing the fusion protein 13GLUCAN1a-CBM2 coded by the nucleotide sequence shown in SEQ ID NO.1, the fusion protein CBM2-13GLUCAN1b coded by the nucleotide sequence shown in SEQ ID NO.2 and a sample to be detected for reaction, and detecting by using an enzyme-labeling instrument;
if the fluorescence intensity is enhanced, determining that the sample to be detected contains beta-1, 3-glucan; if the fluorescence intensity is not enhanced, determining that the sample to be detected does not contain beta-1, 3-glucan.
Advantageous effects
1. The fusion protein 13GLUCAN1a-CBM2 and CBM2-13GLUCAN1b provided by the invention can be specifically combined with beta-1, 3-GLUCAN and generate fluorescence.
2. The fusion protein 13GLUCAN1a-CBM2 and CBM2-13GLUCAN1b provided by the invention are used for detecting beta-1, 3-GLUCAN, the detection method is small in reaction system, short in detection time period, specific in substrate and simple and convenient to operate, the method approaches for detecting the beta-1, 3-GLUCAN are widened, and the fusion protein can also be applied to the field of rapid detection of enzyme activity.
Drawings
FIG. 1 is a graph showing the results of detection of β -1, 3-GLUCAN by fusion protein 13GLUCAN1a-CBM2 and fusion protein CBM2-13GLUCAN1 b.
FIG. 2 is a graph showing the results of the detection of β -1, 3-GLUCAN by the fusion protein 13GLUCAN1a-CBM3 and the fusion protein CBM3-13GLUCAN1 b.
FIG. 3 is a protein electrophoretogram of the fusion protein 13GLUCAN1a-CBM2 of example 5;
in the figure, 13GLUCAN1 a-CBM236.198kDa, the target protein band is circled in black.
FIG. 4 is a protein electrophoretogram of the fusion protein CBM2-13GLUCAN1b of example 5;
in the figure, CBM2-13GLUCAN1b 28.193kDa, and the target protein band is circled in black.
FIG. 5 is a graph showing the results of detection of different substrates in example 6;
in the figure: a1-3, B1-3 and C1-3 are blank controls, and a prepared substrate buffer solution is used as a blank;
a4-6 is 0.2% dextran; a7-9 is 0.2% xylan; b4-6 is 0.2% lignin; b7-9 is 0.2% microcrystalline cellulose; c4-6 is 0.2% hyaluronic acid; c7-9 is 0.2% pullulan.
Detailed Description
The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto.
The examples are not described in detail and are in accordance with the prior art in the field.
Example 1
Gene construction of fusion protein 13GLUCAN1a-CBM2
The N end of the CBM2 gene is connected with a fragment a of a yellow fluorescent protein gene through a Linker to obtain a fusion protein 13GLUCAN1a-CBM2 gene fragment, the nucleotide sequence is shown as SEQ ID NO.1, NdeI and XhoI are selected as restriction enzyme sites, the 13GLUCAN1a-CBM2 gene fragment is inserted into a vector pET20b (+), and a recombinant plasmid pET-20b (+) -13GLUCAN1a-CBM2 is constructed. The constructed plasmid is synthesized by Shanghai Bioengineering company.
Gene construction of fusion protein CBM2-13GLUCAN1b
The C end of the CBM2 gene is connected with a b fragment of a yellow fluorescent protein gene through a Linker to obtain a fusion protein CBM2-13GLUCAN1b gene fragment, the nucleotide sequence is shown as SEQ ID NO.2, NdeI and XhoI are selected as restriction enzyme sites, the CBM2-13GLUCAN1b gene fragment is inserted into a vector pET20b (+), and a recombinant plasmid pET-20b (+) -CBM2-13GLUCAN1b is constructed. The constructed plasmid is synthesized by Shanghai Bioengineering company.
Example 2
Preparation of Escherichia coli competence
(i) Picking a single colony of Escherichia coli (Escherichia coli) BL21(DE3) and inoculating the single colony into an LB culture medium, and culturing at 220r/min and 37 ℃ overnight;
(ii) sucking 0.1mL of bacterial liquid into 10mL of LB culture medium, culturing at 300r/min and 37 ℃ to OD 600 0.6 to 0.8;
(iii) suction 1mLOD 600 Putting the bacterial liquid reaching 0.6-0.8 into a 1.5mL sterile centrifuge tube, centrifuging at 12000r/min for 2min, and completely removing supernatant;
(iv) adding 100 μ L of ice-precooled SSCS (one-step method for rapidly preparing competent cell kit, product of Shanghai Biotechnology engineering company), and suspending thallus lightly to obtain competent cells.
(v) The prepared competent cells were dispensed into 100 μ L each tube and stored at-80 ℃ for future use.
Example 3
13GLUCAN1a-CBM2, CBM2-13GLUCAN1b Gene transformation Escherichia coli (Escherichia coli) BL21(DE3)
The plasmids pET-20b (+) -13GLUCAN1a-CBM2 and pET-20b (+) -CBM2-13GLUCAN1b constructed in example 1 were added with ddH 2 O to make plasmid concentration to 100ng/mL, and the embodiment of the preparation of competent cells for chemical transformation, the cells using 900L LB medium 37 ℃ recovery culture after 1h, centrifugal discard 900L supernatant, using the remaining medium heavy suspension cells, coated on 100 u g/mL LB solid medium, 37 degrees overnight culture, screening with ampicillin resistant transformants. The liquid recovery culture medium comprises the following components per liter:
10g of peptone, 5g of yeast powder, 10g of sodium chloride and the balance of water.
Example 4
Culture and identification of positive recombinant bacteria
The above-mentioned positive recombinant colonies (transformants having ampicillin resistance) were selected, inoculated into a liquid LB medium containing 100. mu.g/mL ampicillin resistance and cultured overnight at 37 ℃, and after completion of the culture, recombinant bacterial DNA was extracted using a kit provided by Shanghai bioengineering Co., Ltd.
The result of agarose gel electrophoresis inspection of the PCR product shows that the size of the 13GLUCAN1a-CBM2 band is about 950bp, which is close to the theoretical value of 963bp, which indicates that the carrier containing the target gene is successfully transferred into the Escherichia coli cell to prepare the Escherichia coli engineering bacteria containing the fusion protein 13GLUCAN1a-CBM2 gene.
The result of agarose gel electrophoresis inspection of the PCR product shows that the size of the CBM2-13GLUCAN1b band is about 700bp, which is close to the theoretical value of 750bp, and the result shows that the carrier containing the target gene is successfully transferred into the escherichia coli cell to prepare the escherichia coli engineering bacteria containing the fusion protein CBM2-13GLUCAN1b gene.
Example 5
Fusion proteins 13GluCAN1a-CBM2, CBM2-13GluCAN1b fermentation test and protein purification.
The engineered Escherichia coli containing the fusion protein 13GLUCAN1a-CBM2 and CBM2-13GLUCAN1b genes prepared in example 4 were inoculated into 100mL LB medium (10 g/L peptone, 5g/L yeast extract, 10g/L NaCl, and the balance water) respectively and cultured at 200rpm and 37 ℃ until OD of the fermentation broth 600 Adding IPTG for induction for 12h between 0.6 and 0.8, and sampling. The bacterial solution was centrifuged to obtain cells, and 0.01M PBS (0.01M PBS preparation method: 8.0g NaCl, 0.2g KCl, 1.44g Na) 2 HPO 4 ,0.24g KH 2 PO 4 Dissolved in 800mLH 2 O, adjusting the pH to 7.4, adding H 2 O to constant volume of 1L), resuspending the thalli, carrying out ultrasonication, centrifuging (10000r/min), and collecting supernatant, namely protein supernatant, wherein a protein electrophoresis chart is shown in figures 3 and 4.
Example 6
Reaction of fusion protein 13GluCAN1a-CBM2, CBM2-13GluCAN1b protein supernatant prepared in example 5 was used.
The fusion protein 13GLUCAN1a-CBM2 and CBM2-13GLUCAN1b can detect fluorescence signals by using an enzyme-labeling instrument (501nm excitation light and 527nm emission light) in a system with beta-1, 3-GLUCAN as a substrate.
The reaction system is as follows:
a200. mu.L reaction was constructed using 80. mu.L of 13GLUCAN1a-CBM2 protein supernatant, 80. mu.L of CBM2-13GLUCAN1b protein supernatant, and 40. mu.L of 0.2% beta-1, 3-GLUCAN at a concentration.
The experimental results are as follows: the fluorescence intensity is obviously increased after the reaction is carried out for 1 hour, the fluorescence intensity is obviously increased within 0-5 hours, and the fluorescence intensity tends to be stable after the reaction is carried out for 5 hours. The fluorescence values of the test sample minus the control sample (control sample without β -1, 3-glucan, all else being identical) were plotted and analyzed, see FIG. 1.
(II) different reaction substrates are used for replacing the beta-1, 3-glucan, and specifically, the mass fraction of the beta-glucan is as follows: 0.2% of pullulan polysaccharide, 0.2% of lignin, 0.2% of microcrystalline cellulose, 0.2% of hyaluronic acid and 0.2% of xylan, and the other conditions are the same.
The results of the experiment, see fig. 5: pullulan, lignin, microcrystalline cellulose, hyaluronic acid and xylan are used as reaction substrates, and no obvious fluorescence enhancement phenomenon exists in a reaction system. Only when the substrate is 0.2 percent of beta-1, 3-GLUCAN, the fluorescence intensity of the reaction system is increased, which indicates that the fusion protein 13GLUCAN1a-CBM2, CBM2-13GLUCAN1b and the beta-1, 3-GLUCAN have good substrate specificity.
Comparative example 1
The fusion protein 13GLUCAN1a-CBM3 and CBM3-13GLUCAN1b are used for detecting beta-1, 3-GLUCAN experiment.
The nucleotide sequence of the encoding gene of the fusion protein 13GLUCAN1a-CBM3 is shown as SEQ ID NO.3, wherein the CBM3 and the CBM2 have similar functions, but have difference with the gene sequence of the CBM 2.
A fusion protein CBM3-13GLUCAN1b encoding gene has a nucleotide sequence shown in SEQ ID NO. 4.
Gene construction of fusion protein 13GLUCAN2a-CBM3
The C end of the CBM3 gene is connected with a fragment a of a yellow fluorescent protein gene through a connecting peptide (Linker) to obtain a fusion protein 13GLUCAN1a-CBM3 gene fragment, the nucleotide sequence is shown in SEQ ID NO.3, NdeI and XhoI are selected as restriction enzyme sites, the 13GLUCAN1a-CBM3 gene fragment is inserted into a vector pET20b (+), and a plasmid of pET-20b (+) -13GLUCAN1a-CBM3 is constructed. The constructed plasmid is synthesized by Shanghai Bioengineering company.
Gene construction of fusion protein CBM3-13GLUCAN1b
The C end of the CBM3 gene is connected with a b fragment of a yellow fluorescent protein gene through a Linker to obtain a fusion protein CBM3-13GLUCAN1b gene fragment, the nucleotide sequence is shown in SEQ ID NO.4, NdeI and XhoI are selected as restriction enzyme sites, the CBM3-13GLUCAN1b gene fragment is inserted into a vector pET20b (+), and a plasmid pET-20b (+) -CBM3-13GLUCAN1b is constructed. The constructed plasmid is synthesized by Shanghai Bioengineering company.
(II) preparation of Escherichia coli competence
(i) Picking a single colony of Escherichia coli (Escherichia coli) BL21(DE3) and inoculating the single colony into an LB culture medium, and culturing at 220r/min and 37 ℃ overnight;
(ii) sucking 0.1mL of bacterial liquid into 10mL of LB culture medium, culturing at 300r/min and 37 ℃ to OD 600 0.6 to 0.8;
(iii) suction 1mLOD 600 Putting the bacterial liquid reaching 0.6-0.8 into a 1.5mL sterile centrifuge tube, centrifuging at 12000r/min for 2min, and completely removing supernatant;
(iv) adding 100 μ L of ice-precooled SSCS (one-step method for rapidly preparing competent cell kit, product of Shanghai Biotechnology engineering company), and suspending thallus lightly to obtain competent cells.
(v) The prepared competent cells were dispensed into 100 μ L each tube and stored at-80 ℃ for future use.
(III) 13GLUCAN1a-CBM3, CBM3-13GLUCAN1b Gene transformation Escherichia coli (Escherichia coli) BL21(DE3)
Adding ddH to the plasmid prepared in the step (one) 2 And O, after the plasmid concentration reaches 100ng/mL, chemically transforming with the competent cells prepared in the step (II), recovering and culturing the obtained cells for 1h at 37 ℃ by using 900 mu LLB culture medium, centrifuging and discarding 900 mu L of supernatant, resuspending the cells by using the rest culture medium, coating the cells on LB solid culture medium containing 100 mu g/mL ampicillin, culturing overnight at 37 ℃, and screening transformants with ampicillin resistance. The liquid recovery culture medium comprises the following components per liter:
10g of peptone, 5g of yeast powder, 10g of sodium chloride and the balance of water.
Culture and Identification of (IV) positive recombinant bacteria
The above-mentioned positive recombinant colonies (transformants having ampicillin resistance) were selected, inoculated into a liquid LB medium containing 100. mu.g/mL ampicillin resistance and cultured overnight at 37 ℃, and after completion of the culture, recombinant bacterial DNA was extracted using a kit provided by Shanghai bioengineering Co., Ltd.
The PCR product is checked by agarose gel electrophoresis, and the result shows that the size of the 13GLUCAN1a-CBM3 band is about 750bp, which is close to the theoretical value of 765bp, which indicates that the carrier containing the target gene is successfully transferred into the escherichia coli cell to prepare the escherichia coli engineering bacteria of the fusion protein 13GLUCAN1a-CBM3 gene.
The result of agarose gel electrophoresis inspection of the PCR product shows that the size of the band of CBM3-13GLUCAN1b is about 550bp, which is close to the theoretical value of 552bp, which indicates that the carrier containing the target gene is successfully transferred into the escherichia coli cell to prepare the escherichia coli engineering bacteria of the fusion protein CBM3-13GLUCAN1b gene.
(V) fusion protein 13GLUCAN a-CBM3, CBM3-13GLUCAN b fermentation test and protein purification.
Respectively inoculating the prepared Escherichia coli engineering bacteria containing fusion protein 13GLUCAN a-CBM3 and CBM3-13GLUCAN b genes into 100mL LB culture medium (10 g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl and the balance of water) at 200rpm and 37 ℃ until fermentation broth OD 600 Adding IPTG for induction for 12h between 0.6 and 0.8, and sampling. Centrifuging the bacterial liquid to obtain thallus, re-suspending the thallus with 0.2M PBS (pH7.4), ultrasonically crushing, centrifuging (10000r/min), and collecting supernatant, i.e. protein supernatant.
Fusion proteins 13GLUCAN1a-CBM3 and CBM3-13GLUCAN1b detect fluorescence signals by using a microplate reader 501nm excitation light and 527nm emission light) in a system with beta-1, 3-GLUCAN as a substrate.
The experimental results show that: the fusion protein 13GLUCAN1a-CBM3 and CBM3-13GLUCAN1b can not detect obvious fluorescence signal difference in a system with beta-1, 3-GLUCAN as a substrate under different proportional concentrations, as shown in figure 2, the structural conflict between CBM3 and 13GLUCAN2a or 13GLUCAN2b is presumed, and the expression activity of the fusion protein is influenced.
In conclusion, the fusion protein 13GLUCAN1a-CBM2 and CBM2-13GLUCAN1b provided by the invention can be specifically combined with beta-1, 3-GLUCAN and generate fluorescence. The fusion protein 13GLUCAN1a-CBM2 and CBM2-13GLUCAN1b provided by the invention are used for detecting beta-1, 3-GLUCAN, the detection method is small in reaction system, short in detection time period, specific in substrate and simple and convenient to operate, the method approaches for detecting the beta-1, 3-GLUCAN are widened, and the fusion protein can also be applied to the field of rapid detection of enzyme activity.
SEQUENCE LISTING
<110> university of Qilu Industrial science
<120> gene construction and application method capable of rapidly detecting beta-1, 3-glucan
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 963
<212> DNA
<213> Artificial sequence
<400> 1
atgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60
gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120
aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180
gtgaccacct tcggctacgg cctgcaatgc ttcgcccgct accccgacca catgaagctg 240
cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300
aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360
aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420
ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacagcgg tggcggtagc 480
ggcggtggca gcggtggcag cataaataac ggcactttcg acgaacctat tgtgaacgat 540
caggccaaca acccggacga atggttcatt tggcaggcgg gagattacgg gatcagcggt 600
gccagggtct ccgattacgg tgtcagggat ggctacgctt atatcacgat agccgatcct 660
ggaactgaca cgtggcatat tcagttcaac cagtggatag gtctttacag aggaaaaacc 720
tacaccattt ctttcaaagc aaaagcggat acaccaagac ctataaatgt gaaaattctg 780
cagaatcacg atccctggac caactatttt gctcaaacgg tgaatctcac agcggactgg 840
cagacgttca cgttcaccta cacgcatcca gacgatgcgg atgaggtcgt tcagatcagt 900
ttcgaactcg gagaaggaac ggcaactacg atttatttcg atgatgtcac ggtgagccct 960
caa 963
<210> 2
<211> 750
<212> DNA
<213> Artificial sequence
<400> 2
atgataaata acggcacttt cgacgaacct attgtgaacg atcaggccaa caacccggac 60
gaatggttca tttggcaggc gggagattac gggatcagcg gtgccagggt ctccgattac 120
ggtgtcaggg atggctacgc ttatatcacg atagccgatc ctggaactga cacgtggcat 180
attcagttca accagtggat aggtctttac agaggaaaaa cctacaccat ttctttcaaa 240
gcaaaagcgg atacaccaag acctataaat gtgaaaattc tgcagaatca cgatccctgg 300
accaactatt ttgctcaaac ggtgaatctc acagcggact ggcagacgtt cacgttcacc 360
tacacgcatc cagacgatgc ggatgaggtc gttcagatca gtttcgaact cggagaagga 420
acggcaacta cgatttattt cgatgatgtc acggtgagcc ctcaaagcgg tggcggtagc 480
ggcggtggca gcggtggcag caagcagaag aacggcatca aggtgaactt caagatccgc 540
cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 600
ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc 660
aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 720
atcactctcg gcatggacga gctgtacaag 750
<210> 3
<211> 765
<212> DNA
<213> Artificial sequence
<400> 3
atgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60
gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120
aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180
gtgaccacct tcggctacgg cctgcaatgc ttcgcccgct accccgacca catgaagctg 240
cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300
aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360
aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420
ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacagcgg tggcggtagc 480
ggcggtggca gcggtggcag cgctgatttc actcaaggag cggacgtctc cggcaacaac 540
gtaactttat ggttcaaatc atcggttaat acgacatggg tggatgtcca ctataaggtg 600
aattccggag tacagcaaaa tgtaaggatg agcttcaacg cgggtgctgc gcgattcgag 660
cacaccattc ttacggccgc ccaagccgag attgagtact ttttcactta caataacggc 720
gtgccagcct acgacaccac aacattcact tatcgaagcg gccaa 765
<210> 4
<211> 552
<212> DNA
<213> Artificial sequence
<400> 4
atggctgatt tcactcaagg agcggacgtc tccggcaaca acgtaacttt atggttcaaa 60
tcatcggtta atacgacatg ggtggatgtc cactataagg tgaattccgg agtacagcaa 120
aatgtaagga tgagcttcaa cgcgggtgct gcgcgattcg agcacaccat tcttacggcc 180
gcccaagccg agattgagta ctttttcact tacaataacg gcgtgccagc ctacgacacc 240
acaacattca cttatcgaag cggccaaagc ggtggcggta gcggcggtgg cagcggtggc 300
agcaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 360
agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 420
ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag 480
cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 540
gagctgtaca ag 552

Claims (10)

1. A fusion protein 13GLUCAN1a-CBM2 coding gene has a nucleotide sequence shown in SEQ ID NO. 1.
2. A recombinant vector, which comprises the nucleotide sequence of the fusion protein 13GLUCAN1a-CBM2 encoding gene of claim 1, as shown in SEQ ID No. 1;
preferably, the recombinant bacterium comprises the nucleotide sequence of the fusion protein 13GLUCAN1a-CBM2 coding gene of claim 1, which is shown as SEQ ID NO. 1.
3. A fusion protein CBM2-13GLUCAN1b encoding gene has a nucleotide sequence shown in SEQ ID NO. 2.
4. A recombinant vector comprising the nucleotide sequence of the encoding gene of the fusion protein CBM2-13GLUCAN1b of claim 3, as shown in SEQ ID No. 2;
preferably, the recombinant bacterium comprises the nucleotide sequence of the encoding gene of the fusion protein CBM2-13GLUCAN1b of claim 3, which is shown as SEQ ID NO. 2.
5. A construction method of Escherichia coli engineering bacteria containing fusion protein 13GLUCAN1a-CBM2 gene is characterized by comprising the following steps:
(1) synthesizing a 13GLUCAN1a-CBM2 gene segment, wherein the nucleotide sequence of the gene segment is shown as SEQ ID NO. 1;
(2) inserting the 13GLUCAN1a-CBM2 gene fragment prepared in the step (1) into a pET-20b (+) vector to obtain a recombinant plasmid pET-20b (+) -13GLUCAN1a-CBM 2;
(3) preparing competent cells of escherichia coli BL21(DE3), carrying out enzyme digestion on the recombinant plasmid pET-20b (+) -13GLUCAN1a-CBM2 prepared in the step (2), transforming the competent cells of escherichia coli BL21(DE3), and screening positive clones to obtain the engineering bacteria of escherichia coli containing the fusion protein 13GLUCAN1a-CBM 2.
6. A construction method of an escherichia coli engineering bacterium containing a fusion protein CBM2-13GLUCAN1b gene is characterized by comprising the following steps:
synthesizing a CBM2-13GLUCAN1b gene segment, wherein the nucleotide sequence of the CBM2-13GLUCAN1b gene segment is shown as SEQ ID NO. 2;
secondly, inserting the CBM2-13GLUCAN1b gene fragment prepared in the step I into a pET-20b (+) vector to obtain a recombinant plasmid pET-20b (+) -CBM2-13GLUCAN1 b;
preparing competent cells of escherichia coli BL21(DE3), carrying out enzyme digestion on the recombinant plasmid pET-20b (+) -CBM2-13GLUCAN1b prepared in the step II, transforming the competent cells of escherichia coli BL21(DE3), and screening positive clones to obtain the engineering bacteria of escherichia coli containing the fusion protein CBM2-13GLUCAN1 b.
7. The method of claim 5 or 6, wherein the positive clones selected in step (3) or step (iii) are selected by coating transformed cells on LB solid medium containing 100. mu.g/mL ampicillin, culturing at 37 ℃, selecting a single colony, inoculating it into LB liquid medium containing 100. mu.g/mL ampicillin, culturing overnight at 37 ℃, verifying by PCR to obtain positive clones of the target gene band, and after sequencing, retaining the strain with correct sequencing result as the target strain.
8. The use of the engineered Escherichia coli containing the fusion protein 13GLUCAN1a-CBM2 gene constructed by the method of claim 5 in the production of fusion protein 13GLUCAN1a-CBM 2;
preferably, the application of the engineering bacterium of Escherichia coli containing the CBM2-13GLUCAN1b gene constructed by the method of claim 6 in the production of the CBM2-13GLUCAN1b fusion protein.
9. The fusion protein 13GLUCAN1a-CBM2 coded by the nucleotide sequence shown in SEQ ID NO.1 and the fusion protein CBM2-13GLUCAN1b coded by the nucleotide sequence shown in SEQ ID NO.2 are applied to the detection of beta-1, 3-GLUCAN.
10. A method for detecting β -1, 3-glucan, comprising the steps of:
mixing the fusion protein 13GLUCAN1a-CBM2 coded by the nucleotide sequence shown in SEQ ID NO.1, the fusion protein CBM2-13GLUCAN1b coded by the nucleotide sequence shown in SEQ ID NO.2 and a sample to be detected for reaction, and detecting by using an enzyme-labeling instrument;
if the fluorescence intensity is enhanced, determining that the sample to be detected contains beta-1, 3-glucan; if the fluorescence intensity is not enhanced, determining that the sample to be detected does not contain beta-1, 3-glucan.
CN202210743979.6A 2022-06-27 2022-06-27 Gene construction and application method capable of rapidly detecting beta-1, 3-glucan Pending CN114958885A (en)

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CN104004098A (en) * 2014-05-29 2014-08-27 清华大学 Vector composition for indicating active state of Wnt signal in cell by using BiFC (Bimolecular Fluorescence Complementation) and application of vector composition
WO2022050316A1 (en) * 2020-09-07 2022-03-10 東栄新薬株式会社 METHOD FOR DETECTING AND QUANTIFYING β-1,6-BRANCHED β-1,3-GLUCAN OR β-1,3-GLUCAN, AND KIT FOR DETECTING AND QUANTIFYING SAME

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Publication number Priority date Publication date Assignee Title
US20030096329A1 (en) * 2001-11-16 2003-05-22 Seikagaku Corporation (1-3)-beta-D-glucan binding domain protein, measuring method using the substance and assay kit
CN104004098A (en) * 2014-05-29 2014-08-27 清华大学 Vector composition for indicating active state of Wnt signal in cell by using BiFC (Bimolecular Fluorescence Complementation) and application of vector composition
WO2022050316A1 (en) * 2020-09-07 2022-03-10 東栄新薬株式会社 METHOD FOR DETECTING AND QUANTIFYING β-1,6-BRANCHED β-1,3-GLUCAN OR β-1,3-GLUCAN, AND KIT FOR DETECTING AND QUANTIFYING SAME

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